Oligonucleotide compositions and methods of use thereof

Abstract

The present disclosure provides, inter alia, designed APOC3 oligonucleotides, compositions, and methods thereof. In some embodiments, the provided oligonucleotide compositions provide improved single stranded RNA interference and / or RNase H mediated knock-down. The present disclosure encompasses, among other things, the recognition of structural elements of oligonucleotides, such as base sequences, chemical modifications (e.g., modifications of sugars, bases and / or inter-nucleotide linkages) or patterns thereof, conjugation to additional chemical moieties and / or stereochemistry [e.g., stereochemistry of the backbone chiral center (chiral inter-nucleotide linkages)] and / or patterns thereof, or combinations thereof. The oligonucleotide has significant influence on properties and activity of the oligonucleotide, such as RNA interference (RNAi) activity, stability, delivery and the like. In some embodiments, the disclosure provides methods of treating disease using the provided oligonucleotide compositions (e.g., in RNA interference and / or RNase H mediated knock-down).

Description

[0001] This application is a divisional application of a Chinese patent application with application number 201880049562.1, filing date of June 1, 2018, and invention title of "Oligonucleotide Compositions and Methods of Use Thereof".

[0002] Cross - reference to related applications

[0003] This application claims priority to U.S. Provisional Application No. 62 / 514,769, filed on June 2, 2017, and U.S. Provisional Application No. 62 / 670,702, filed on May 11, 2018, each of which is incorporated herein by reference in its entirety. Background of the Invention

[0004] Oligonucleotides targeting APOC3 (APOC3 oligonucleotides) can be used in various applications, such as therapeutic applications. The use of naturally occurring nucleic acids (e.g., unmodified DNA or RNA) can be limited, for example, by their susceptibility to endonucleases and exonucleases. Summary of the Invention

[0005] This disclosure particularly encompasses the recognition that controlling the structural elements of APOC3 oligonucleotides, such as chemical modifications (e.g., modifications of sugars, bases, and / or internucleotide linkages) or their patterns, changes in stereochemistry (e.g., the stereochemistry of backbone chiral internucleotide linkages) or their patterns, and / or conjugation with additional chemical moieties (e.g., lipid moieties, targeting moieties, carbohydrate moieties, moieties that bind to the asialoglycoprotein receptor or ASGPR, such as GalNAc moieties), etc., can have a significant impact on the properties and / or activities of APOC3 oligonucleotides. In some embodiments, the properties and / or activities include, but are not limited to, participating in guiding the reduction of the expression, activity, or level of the APOC3 gene or its gene product mediated, for example, by RNA interference (RNAi interference), single - stranded RNA interference (ssRNAi), RNase H - mediated knockdown, steric hindrance of translation, etc.

[0006] It should be understood that the foregoing general description and the following detailed description are merely exemplary and explanatory and do not limit the claimed invention. Detailed Description of the Invention

[0008] The present invention can be more readily understood by reference to the following detailed description of exemplary embodiments of the invention and the examples included therein.

[0009] It should be understood that the present invention is not limited to the specified preparation and synthesis methods described, which can of course vary. It should also be understood that the terms used herein are for the purpose of describing particular embodiments only and are not intended to be limiting.

[0010] In some embodiments, the present disclosure encompasses the recognition that stereochemistry, particularly the stereochemistry of backbone chiral centers, can unexpectedly improve the properties of APOC3 oligonucleotides. Contrary to many previous observations where some structural elements that increase stability can also decrease activity, such as RNA interference, the present disclosure demonstrates that control of stereochemistry can surprisingly increase stability without significantly reducing activity.

[0011] In some embodiments, the present disclosure provides oligonucleotides having certain 5′-terminal structures.

[0012] In some embodiments, the present disclosure provides 5′-terminal structures that can provide oligonucleotides having high biological activity (e.g., RNAi activity) when used in accordance with the present disclosure.

[0013] In some embodiments, the present disclosure encompasses the recognition that various additional chemical moieties, such as lipid moieties and / or carbohydrate moieties, can improve one or more APOC3 oligonucleotide properties, such as knockdown of an APOC3 target gene or its gene product, when incorporated into the oligonucleotide. In some embodiments, the additional chemical moieties are optional. In some embodiments, the APOC3 oligonucleotide can comprise more than one additional chemical moiety. In some embodiments, the APOC3 oligonucleotide can comprise two or more additional chemical moieties, where the additional chemical moieties are the same or different, or belong to the same class (e.g., targeting moiety, carbohydrate moiety, moiety that binds to ASPGR, lipid moiety, etc.) or do not belong to the same class. In some embodiments, certain additional chemical moieties facilitate delivery of the oligonucleotide to a desired cell, tissue, and / or organ. In some embodiments, certain additional chemical moieties facilitate internalization of the oligonucleotide and / or increase the stability of the oligonucleotide.

[0014] In some embodiments, the present disclosure demonstrates that certain provided structural elements, techniques, and / or features are particularly useful for APOC3 oligonucleotides (e.g., RNAi agents) that participate in and / or direct the RNAi mechanism. However, in any case, the teachings of the present disclosure are not limited to oligonucleotides that participate or act through any particular mechanism. In some embodiments, the present disclosure relates to any oligonucleotide that can act through any mechanism and that includes any sequence, structure, or format (or portions thereof) described herein. In some embodiments, the present disclosure provides APOC3 oligonucleotides that can act through any mechanism and that include any sequence, structure, or format (or portions thereof) described herein, including but not limited to any 5′-terminal structure; 5′-terminal region; first region (including but not limited to the seed region); second region (including but not limited to the post-seed region); and 3′-terminal region (which can be a 3′-terminal dinucleotide and / or 3′-terminal cap); optional additional chemical moieties (including but not limited to targeting moieties, carbohydrate moieties, APGR-binding moieties, and lipid moieties); stereochemistry or stereochemical patterns; modifications or modification patterns; internucleotide linkages or internucleotide linkage patterns; modifications of the sugar or sugar modification patterns; modifications of the base or base modification patterns. In some embodiments, the provided oligonucleotides can participate in (e.g., direct) the RNAi mechanism. In some embodiments, the provided oligonucleotides can participate in the RNase H (ribonuclease H) mechanism. In some embodiments, the provided oligonucleotides can act as translation inhibitors (e.g., can provide steric hindrance to translation). In some embodiments, the provided oligonucleotides can be therapeutic.

[0015] In some embodiments, the APOC3 target sequence is a sequence that binds to an APOC3 oligonucleotide as described herein. In many embodiments, the target sequence is identical or precisely complementary to the provided oligonucleotide or the sequence of contiguous residues therein (e.g., the provided oligonucleotide includes a targeting binding sequence that is identical or precisely complementary to the target sequence). In some embodiments, the targeting binding sequence is precisely complementary to the target sequence of a transcript (e.g., pre-mRNA, mRNA, etc.). The targeting binding sequence / target sequence can have various lengths to provide oligonucleotides with desired activities and / or properties. In some embodiments, the targeting binding sequence / target sequence includes 5 - 50 bases. In some embodiments, a small number of differences / mismatches are tolerated between the APOC3 oligonucleotide (relevant portion) and its target sequence (including but not limited to the 5′- and / or 3′-terminal regions of the target and / or oligonucleotide sequence). In many embodiments, the target sequence is present in a transcript (e.g., mRNA and / or pre-mRNA) produced by the target gene.

[0016] Unless otherwise indicated, all sequences (including but not limited to base sequences and chemical, modified, and / or stereochemical patterns) are presented in the 5′ to 3′ order.

[0017] In some embodiments, the present disclosure provides compositions and methods related to APOC3 oligonucleotides that are specific for a target and have or comprise the base sequence of any oligonucleotide disclosed herein or a region of at least 15 contiguous nucleotides of the base sequence of any oligonucleotide disclosed herein, wherein the first nucleotide of the base sequence or the first nucleotide of the at least 15 contiguous nucleotides can optionally be replaced by T or DNA T. In some embodiments, the oligonucleotide is capable of directing ssRNAi.

[0018] In some embodiments, the present disclosure provides an APOC3 oligonucleotide composition comprising a first plurality of oligonucleotides having a common base sequence and comprising one or more internucleotide linkages, sugar, and / or base modifications.

[0019] In some embodiments, the nucleotide is a natural nucleotide. In some embodiments, the nucleotide is a modified nucleotide. In some embodiments, the nucleotide is a nucleotide analogue. In some embodiments, the base is a modified base. In some embodiments, the base is a protected nucleobase, such as a protected nucleobase used in oligonucleotide synthesis. In some embodiments, the base is a base analogue. In some embodiments, the sugar is a modified sugar. In some embodiments, the sugar is a sugar analogue. In some embodiments, the internucleotide linkage is a modified internucleotide linkage. In some embodiments, the nucleotide comprises a base, a sugar, and an internucleotide linkage, wherein each of the base, the sugar, and the internucleotide linkage is independently and optionally naturally occurring or non-naturally occurring. In some embodiments, the nucleoside comprises a base and a sugar, wherein each of the base and the sugar is independently and optionally naturally occurring or non-naturally occurring. Non-limiting examples of nucleotides include DNA (2′-deoxy) and RNA (2′-OH) nucleotides; and those nucleotides comprising one or more modifications at the base, sugar, and / or internucleotide linkage. Non-limiting examples of sugars include ribose and deoxyribose; and ribose and deoxyribose having 2′-modifications, the 2′-modifications including but not limited to 2′-F, LNA, 2′-OMe, and 2′-MOE modifications. In some embodiments, the internucleotide linkage can have the structure of Formula I as disclosed herein. In some embodiments, the internucleotide linkage is a moiety that does not contain phosphorus but is used to link two natural or non-natural sugars.

[0020] In some embodiments, the present disclosure provides a chirally controlled APOC3 oligonucleotide composition that directs a greater reduction in the expression, activity, and / or level of the APOC3 gene or its gene product, single-stranded RNA interference, and / or RNase H-mediated knockdown, when compared to a reference condition (e.g., absence of the composition or absence of a reference composition (e.g., a stereorandom composition of oligonucleotides having the same base sequence and chemical modifications)).

[0021] In some embodiments, an APOC3 oligonucleotide composition comprising a plurality of oligonucleotides is stereorandom because the plurality of oligonucleotides do not share a common stereochemistry at any chiral internucleotide linkage. In some embodiments, an APOC3 oligonucleotide composition comprising a plurality of oligonucleotides is chirally controlled because the plurality of oligonucleotides share a common stereochemistry at one or more chiral internucleotide linkages. In some embodiments, a chirally controlled APOC3 oligonucleotide composition comprising a first plurality of oligonucleotides is less sensitive to endonucleases and exonucleases relative to a stereorandom APOC3 oligonucleotide composition comprising the first plurality of oligonucleotides.

[0022] In some embodiments, the composition comprises a multimer of two or more of: a first plurality of APOC3 oligonucleotides and / or a second plurality of oligonucleotides, wherein the first and second plurality of oligonucleotides can independently direct knockdown of the same or different targets by RNA interference and / or RNase H-mediated knockdown.

[0023] In some embodiments, an APOC3 oligonucleotide composition comprising a plurality of oligonucleotides (e.g., a first plurality of oligonucleotides) is chirally controlled because the plurality of oligonucleotides independently share a common stereochemistry at one or more chiral internucleotide linkages. In some embodiments, the plurality of oligonucleotides share a common stereochemical configuration at 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50 or more chiral internucleotide linkages, each of which is independently Rp or Sp. In some embodiments, the plurality of oligonucleotides share a common stereochemical configuration at each chiral internucleotide linkage. In some embodiments, a chiral internucleotide linkage is referred to as a chirally controlled internucleotide linkage when a predetermined level of the oligonucleotides in the composition share a common stereochemical configuration (independently Rp or Sp).

[0024] In some embodiments, at least 5 internucleotide linkages are chirally controlled; in some embodiments, at least 10 internucleotide linkages are chirally controlled; in some embodiments, at least 15 internucleotide linkages are chirally controlled; in some embodiments, each chiral internucleotide linkage is chirally controlled.

[0025] In some embodiments, the present disclosure provides an APOC3 oligonucleotide composition comprising a first plurality of APOC3 oligonucleotides that share:

[0026] 1) a common base sequence;

[0027] 2) a common backbone linkage pattern; and

[0028] 3) a common backbone chiral center pattern, wherein the composition is a substantially pure preparation of a single oligonucleotide, because a predetermined level of the oligonucleotides in the composition have a common base sequence and length, a common backbone linkage pattern, and a common backbone chiral center pattern.

[0029] In some embodiments, the common backbone chiral center pattern includes at least one internucleotide linkage comprising a chiral center with chirally controlled.

[0030] In some embodiments, the level of oligonucleotide and / or diastereomeric purity can be determined by analytical methods (such as chromatography, spectrophotometry, spectroscopy, or any combination thereof).

[0031] The present disclosure particularly encompasses the recognition that a stereorandom APOC3 oligonucleotide preparation contains multiple different chemical entities that differ from each other in terms of the stereochemical structure (or stereochemistry) of various backbone chiral centers within the oligonucleotide chain, for example. Without controlling the stereochemistry of the backbone chiral centers, a stereorandom oligonucleotide preparation provides an uncontrolled composition that contains an undefined level of oligonucleotide stereoisomers. Even though these stereoisomers may have the same base sequence and / or chemical modifications, they are different chemical entities at least because of their different backbone stereochemistry, and as demonstrated herein they can have different properties, such as sensitivity to nucleases, activity, distribution, etc. In some embodiments, a particular stereoisomer can be defined, for example, by its base sequence, its length, its backbone linkage pattern, and its backbone chiral center pattern. In some embodiments, the present disclosure demonstrates that the improvements in properties and activities achieved by controlling the stereochemistry within APOC3 oligonucleotides can be comparable or even better than those achieved by using chemical modifications.

[0032] In some embodiments, the APOC3 oligonucleotide comprises a 5′-terminal region, a seed region, a post-seed region, and a 3′-terminal region in a 5′ to 3′ order, optionally further comprising additional chemical moieties.

[0033] In some embodiments, the 5′-terminal region is the entire portion of the APOC3 oligonucleotide that is 5′ to the seed region. In some embodiments, the 3′-terminal region is the entire portion of the APOC3 oligonucleotide that is 3′ to the post-seed region.

[0034] In some embodiments, the 5′-terminal structure is a 5′-terminal group.

[0035] In some embodiments, the 5′-terminal structure comprises a 5′-terminal group.

[0036] In some embodiments, the provided oligonucleotide may comprise a 5′-terminal region, a 5′-terminal structure, a 5′-terminal group, a 5′-terminal nucleoside, or a 5′-terminal nucleotide as described herein or known in the art.

[0037] In some embodiments, the 5′-terminal structure, the 5′-terminal region, the 5′-nucleotide moiety, the seed region, the post-seed region, the 3′-terminal dinucleotide, and / or the 3′-terminal cap independently have any structure as described herein or known in the art. In some embodiments, any structure of the 5′-terminal as described herein or known in the art, and / or any structure of the 5′-nucleotide moiety as described herein or known in the art, and / or any structure of the seed region as described herein or known in the art, and / or any structure of the post-seed region as described herein or known in the art, and / or any structure of the 3′-terminal dinucleotide as described herein or known in the art, and / or any structure of the 3′-terminal cap as described herein or known in the art may be combined.

[0038] In some embodiments, the provided oligonucleotide comprises one or more blocks. In some embodiments, the provided oligonucleotide comprises one or more blocks, wherein the block comprises one or more consecutive nucleosides, and / or nucleotides, and / or sugars or bases, and / or internucleotide linkages. In some embodiments, the block encompasses the entire seed region or a portion thereof. In some embodiments, the block encompasses the entire post-seed region or a portion thereof.

[0039] In some embodiments, the provided oligonucleotide is a block polymer.

[0040] In some embodiments, the provided oligonucleotides are alternating polymers comprising alternating blocks. In some embodiments, the block polymers or alternating polymers can be defined by chemical modifications (including the presence or absence thereof) (e.g., base modifications, sugar modifications, internucleotide linkage modifications, stereochemistry, etc.) or patterns thereof.

[0041] In some embodiments, the provided oligonucleotides comprise one or more sugar modifications. In some embodiments, the sugar modification is at the 2′-position. In some embodiments, the sugar modification is selected from: 2′-F, 2′-OMe, and 2′-MOE. 2′-F is also known as 2′-fluoro. 2′-OMe is also known as 2′-O-methyl. 2′-MOE is also known as 2′-methoxyethyl or MOE.

[0042] In some embodiments, the APOC3 oligonucleotide comprises only two 2′-F. In some embodiments, the APOC3 oligonucleotide comprises only two 2′-F, wherein the two nucleotides are at positions 2 and 14.

[0043] In some embodiments, the APOC3 oligonucleotide comprises only two 2′-F, wherein the two nucleotides are at positions 2 and 14, and wherein the first nucleotide is 2′-deoxy.

[0044] In some embodiments, the APOC3 oligonucleotide comprises only two 2′-F, wherein the two nucleotides are at positions 2 and 14, and wherein the first nucleotide is 2′-deoxy T.

[0045] In some embodiments, the APOC3 oligonucleotide comprises only two 2′-F, wherein the two nucleotides are at positions 2 and 14, and wherein the first nucleotide is 2′-deoxy, and the 5′-terminal structure is -OH.

[0046] In some embodiments, the APOC3 oligonucleotide comprises only two 2′-F, wherein the two nucleotides are at positions 2 and 14, and wherein the first nucleotide is 2′-deoxy T, and the 5′-terminal structure is -OH.

[0047] In some embodiments herein, with respect to the APOC3 oligonucleotide, “first” (e.g., first nucleotide) refers to the 5′-end of the oligonucleotide, and “last” or “terminal” (e.g., last nucleotide or terminal nucleotide) refers to the 3′-end.

[0048] In some embodiments, the provided oligonucleotides comprise sugars with specific modifications that alternate with sugars that are unmodified or have different modifications. In some embodiments, the sugars with specific modifications occur in one or more blocks.

[0049] In some embodiments, the provided oligonucleotides comprise one or more blocks, the one or more blocks comprising sugars having a particular 2'-modification, which alternate with sugars that are independently unmodified or have a different modification. In some embodiments, the provided oligonucleotides comprise one or more blocks, the one or more blocks comprising sugars having a 2'-F modification, which alternate with sugars that are independently unmodified or have a different modification. In some embodiments, the provided oligonucleotides comprise one or more blocks, the one or more blocks comprising sugars having a 2'-OMe modification, which alternate with sugars that are independently unmodified or have a different modification. In some embodiments, the provided oligonucleotides comprise one or more blocks, the one or more blocks comprising sugars having a 2'-OMe modification, which alternate with sugars having a 2'-F modification.

[0050] In some embodiments, the sugar block has or comprises a 2′-modified pattern of any of the following: ff, fffm, fffmm, fffmmm, fffmmmm, fffmmmmm, fffmmmmmm, fffmmmmmmf, fffmmmmmmff, fffmmmmmmffm, fffmmmmmmffmm, fffmmmmmmffmmf, fffmmmmmmffmmfm, fffmmmmmmffmmfmf, fffmmmmmmffmmfmfm, fffmmmmmmffmmfmfmf, fffmmmmmmffmmfmfmfm, fffmmmmmmffmmfmfmfmm, fffmmmmmmffmmfmfmfmmm, ffmmffmm, ffmmmmmmffmmfmfmfmmm, fmfmfmfmfmfmfm, fmfmfmfmfmfmfmf, fmfmfmfmfmfmfmfm, fmfmfmfmfmfmfmfmf, fmfmfmfmfmfmfmfmfm, fmfmfmfmfmfmfmfmfmf, fmfmfmfmfmfmfmfmfmfm, fmfmfmfmfmfmfmfmfmfmm, fmfmfmfmfmfmfmfmfmm, fmfmfmfmfmfmfmfmm, fmfmfmfmfmfmfmm, fmfmfmfmfmfmmm, fmmffmm, fmmmmmmffmmfmfmfmmm, mff, mffm, mffmf, mffmff, mffmffm, mffmmffmm, mfmfm, mfmfmfmfmfffmfmfmfmmm, mfmfmfmfmfmfmfm, mfmfmfmfmfmfmfmfmfmm, mfmfmfmfmfmfmfmfmfmmm, mfmfmfmfmfmfmfmfmm, mfmfmfmfmfmfmfmm, mfmfmfmfmfmfmm, mfmfmfmfmfmfmmm, mfmfmfmfmfmmm, mfmfmfmfmfmmmfm, mfmfmfmfmfmmmmm, mfmfmfmfmmm, mfmfmfmfmmmfmfm, mfmfmfmfmmmfmmm, mfmfmfmfmmmmmfm, mfmfmfmmm, mfmfmfmmmfmfmfm, mfmfmfmmmfmfmmm, mfmfmfmmmfmfmmmfm, mfmfmfmmmmmfmfm, mfmfmmm, mfmfmmmfmfmfmfm, mfmfmmmfmfmfmmm, mfmfmmmfmfmmmfm, mfmfmmmfmmmfmfm,mfmfmmmmmfmfmfm, mfmmm, mfmmmfmfmfmfmfm, mfmmmfmfmfmfmmm, mfmmmfmfmfmmmfm, mfmmmfmfmmmfmfm, mfmmmfmmmfmfmfm, mfmmmfmmmfmfmfm, mfmmmmmfmfmfmfm, mmffm, mmffmm, mmffmm, mmffmmf, mmffmmff, mmffmmffm, mmffmmffmm, mmffmmfmfmfmmm, mmm, mmmffmmfmfmfmmm, mmmfmfmfmfmfmfm, mmmfmfmfmfmfmmm, mmmfmfmfmfmmmfm, mmmfmfmfmmmfmfm, mmmfmfmmmfmfmfm, mmmfmmmfmfmfmfm, mmmmmmffmmfmfmfmmm, mmm, mmmm, mmmmmm, mmmmmmffmmfmfmfmmm, mmmmmmfmfmfmfmfm, mmmmmmm, mmmmmmmffmmfmfmfmmm, mfmf, mfmf, mfmfmf, fmfm, fmfmfm, fmfmfmf, dfdf, dfdfdf, dfdfdfdf, fdfd, fdfdfd, fdfdfdfd, dfdfmfmf, dfmfmf, mfdfmf or dfmfdf, where m represents 2′-OMe, f represents 2′-F, and d represents no substitution at the 2′-position. In some embodiments, the seed region and / or the post-seed region may contain a sugar-modified block.

[0051] In some embodiments, the block is a stereochemical block. In some embodiments, the block is an Rp block because each internucleotide linkage of the block is Rp. In some embodiments, the seed region-block is an Rp block. In some embodiments, the post-seed region-block is an Rp block. In some embodiments, the block is an Sp block because each internucleotide linkage of the block is Sp. In some embodiments, the seed region-block is an Sp block. In some embodiments, the post-seed region-block is an Sp block. In some embodiments, the provided oligonucleotide contains both Rp blocks and Sp blocks. In some embodiments, the provided oligonucleotide contains one or more Rp blocks but does not contain Sp blocks. In some embodiments, the provided oligonucleotide contains one or more Sp blocks but does not contain Rp blocks. In some embodiments, the provided oligonucleotide contains one or more PO blocks, where each internucleotide linkage of the block is a native phosphoester linkage.

[0052] In some embodiments, the seed region-block is an Sp block, wherein each sugar moiety comprises a 2′-F modification. In some embodiments, the seed region-block is an Sp block, wherein each internucleotide linkage is a modified internucleotide linkage and each sugar moiety comprises a 2′-F modification. In some embodiments, the seed region-block is an Sp block, wherein each internucleotide linkage is a phosphorothioate linkage and each sugar moiety comprises a 2′-F modification. In some embodiments, the seed region-block comprises 4 or more nucleoside units. In some embodiments, the nucleoside unit is a nucleoside. In some embodiments, the seed region-block comprises 5 or more nucleoside units. In some embodiments, the seed region-block comprises 6 or more nucleoside units. In some embodiments, the seed region-block comprises 7 or more nucleoside units. In some embodiments, the post-seed region-block is an Sp block, wherein each sugar moiety comprises a 2′-F modification. In some embodiments, the post-seed region-block is an Sp block, wherein each internucleotide linkage is a modified internucleotide linkage and each sugar moiety comprises a 2′-F modification. In some embodiments, the post-seed region-block is an Sp block, wherein each internucleotide linkage is a phosphorothioate linkage and each sugar moiety comprises a 2′-F modification. In some embodiments, the post-seed region-block comprises 4 or more nucleoside units. In some embodiments, the post-seed region-block comprises 5 or more nucleoside units. In some embodiments, the post-seed region-block comprises 6 or more nucleoside units. In some embodiments, the post-seed region-block comprises 7 or more nucleoside units. In some embodiments, the seed region and / or the post-seed region can comprise blocks. In some embodiments, the seed region and / or the post-seed region comprise stereochemical blocks.

[0053] In some embodiments, the present disclosure provides an APOC3 oligonucleotide composition comprising a first plurality of oligonucleotides, the first plurality of oligonucleotides:

[0054] 1) have a common base sequence; and

[0055] 2) comprise one or more modified sugar moieties and modified internucleotide linkages.

[0056] In some embodiments, the present disclosure provides an APOC3 oligonucleotide composition comprising a first plurality of oligonucleotides capable of directing single-stranded RNA interference, the first plurality of oligonucleotides:

[0057] 1) have a common base sequence complementary to a target sequence in a transcript; and

[0058] 2) comprise one or more modified sugar moieties and modified internucleotide linkages.

[0059] In some embodiments, the reference condition is the absence of a composition. In some embodiments, the reference condition is the presence of a reference composition. Exemplary reference compositions comprising a plurality of reference oligonucleotides are described in detail in the present disclosure. In some embodiments, the plurality of reference oligonucleotides have different structural elements (chemical modifications, stereochemistry, etc.) compared to the first plurality of oligonucleotides in the provided composition. In some embodiments, the oligonucleotide composition comprising the first plurality of oligonucleotides is chirally controlled because the first plurality of oligonucleotides comprise one or more chirally controlled internucleotide linkages. In some embodiments, the oligonucleotide composition comprising the first plurality of oligonucleotides is chirally controlled because the first plurality of oligonucleotides comprise 1-20 chirally controlled internucleotide linkages. In some embodiments, the first plurality of oligonucleotides comprise 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 chirally controlled internucleotide linkages. In some embodiments, the reference composition is a stereorandom article of oligonucleotides having the same chemical modification. In some embodiments, the reference composition is a mixture of stereoisomers, while the provided composition is a single-stranded RNAi agent of one stereoisomer. In some embodiments, in the provided composition, the plurality of reference oligonucleotides have the same base sequence as the first plurality of oligonucleotides. In some embodiments, in the provided composition, the plurality of reference oligonucleotides have the same chemical modification as the first plurality of oligonucleotides. In some embodiments, in the provided composition, the plurality of reference oligonucleotides have the same sugar modification as the first plurality of oligonucleotides. In some embodiments, in the provided composition, the plurality of reference oligonucleotides have the same base modification as the first plurality of oligonucleotides. In some embodiments, in the provided composition, the plurality of reference oligonucleotides have the same internucleotide linkage modification as the first plurality of oligonucleotides. In some embodiments, in the provided composition, the plurality of reference oligonucleotides have the same base sequence and the same chemical modification as the first plurality of oligonucleotides. In some embodiments, in the provided composition, the plurality of reference oligonucleotides have the same stereochemistry as the first plurality of oligonucleotides, but have different chemical modifications, such as base modifications, sugar modifications, internucleotide linkage modifications, etc.

[0060] In some embodiments, the present disclosure provides a composition comprising an APOC3 oligonucleotide, wherein the oligonucleotide is complementary or substantially complementary to a target RNA sequence, has a length of from about 15 to about 49 nucleotides in total, and wherein the oligonucleotide comprises at least one unnatural base, sugar, and / or internucleotide linkage.

[0061] In some embodiments, the present disclosure provides an APOC3 oligonucleotide composition comprising a single-stranded RNAi agent, wherein the single-stranded RNAi agent is complementary or substantially complementary to a target RNA sequence, has a length of from about 15 to about 49 nucleotides in total, and is capable of directing target-specific RNA interference, and wherein the single-stranded RNAi agent comprises at least one unnatural base, sugar, and / or internucleotide linkage.

[0062] In some embodiments, the length is from 15 to 49, about 17 to about 49, 17 to 49, about 19 to about 29, 19 to 29, about 19 to about 25, 19 to 25, about 19 to about 23, or 19 to 23 nucleotides in total.

[0063] In some embodiments, the present disclosure provides an APOC3 oligonucleotide composition comprising a first plurality of oligonucleotides, the first plurality of oligonucleotides:

[0064] 1) have a common base sequence that is complementary or substantially complementary to a target sequence in a transcript; and

[0065] 2) comprise one or more modified sugar moieties and modified internucleotide linkages,

[0066] wherein the oligonucleotide composition is characterized in that when contacted with the transcript, knockdown of the transcript is improved compared to the knockdown observed under reference conditions, the reference conditions being selected from the group consisting of the absence of the composition, the presence of a reference composition, and combinations thereof.

[0067] In some embodiments, the present disclosure provides an APOC3 oligonucleotide composition comprising a first plurality of oligonucleotides capable of directing single-stranded RNA interference, the first plurality of oligonucleotides:

[0068] 1) have a common base sequence that is complementary to a target sequence in a transcript; and

[0069] 2) comprise one or more modified sugar moieties and modified internucleotide linkages,

[0070] wherein the oligonucleotide composition is characterized in that when contacted with the transcript in an RNA interference system, RNAi-mediated knockdown of the transcript is improved compared to the knockdown observed under reference conditions, the reference conditions being selected from the group consisting of the absence of the composition, the presence of a reference composition, and combinations thereof.

[0071] In some embodiments, the present disclosure provides an APOC3 oligonucleotide composition comprising a first plurality of oligonucleotides, wherein the first plurality of oligonucleotides belong to a specific oligonucleotide type, which is defined by:

[0072] 1) base sequence;

[0073] 2) Main chain linkage pattern;

[0074] 3) Main chain chiral center pattern; and

[0075] 4) Main chain phosphorus modification pattern.

[0076] In some embodiments, the present disclosure provides an APOC3 oligonucleotide composition comprising a first plurality of oligonucleotides capable of directing single-stranded RNA interference, wherein the first plurality of oligonucleotides belong to a specific oligonucleotide type, which is defined by:

[0077] 1) Base sequence;

[0078] 2) Main chain linkage pattern;

[0079] 3) Main chain chiral center pattern; and

[0080] 4) Main chain phosphorus modification pattern.

[0081] In some embodiments, the present disclosure provides an APOC3 oligonucleotide composition comprising a first plurality of oligonucleotides of an APOC3 oligonucleotide type, wherein the oligonucleotide type is defined by:

[0082] 1) Base sequence;

[0083] 2) Main chain linkage pattern;

[0084] 3) Main chain chiral center pattern; and

[0085] 4) Main chain phosphorus modification pattern,

[0086] The composition is chirally controlled because it is enriched in oligonucleotides of the specific oligonucleotide type relative to a substantially racemic product of oligonucleotides having the same base sequence.

[0087] The oligonucleotide composition is characterized in that when it contacts a transcript, the knockdown of the transcript is improved compared to the knockdown observed under reference conditions, which are selected from the group consisting of the absence of the composition, the presence of a reference composition, and combinations thereof.

[0088] In some embodiments, the present disclosure provides an APOC3 oligonucleotide composition comprising a first plurality of oligonucleotides capable of directing single-stranded RNA interference and belonging to an APOC3 oligonucleotide type, wherein the oligonucleotide type is defined by:

[0089] 1) Base sequence;

[0090] 2) Main chain linkage pattern;

[0091] 3) The style of the chiral center on the main chain; and

[0092] 4) The style of the phosphorus modification on the main chain,

[0093] The composition is chirally controlled because it is enriched in oligonucleotides of the specific oligonucleotide type relative to a substantially racemic product of oligonucleotides having the same base sequence.

[0094] The oligonucleotide composition is characterized in that when it contacts a transcript in an RNA interference system, the RNAi-mediated knockdown of the transcript is improved compared to the knockdown observed under reference conditions, the reference conditions being selected from the group consisting of the absence of the composition, the presence of a reference composition, and combinations thereof.

[0095] In some embodiments, the provided oligonucleotides have any format shown in FIG. 1, or any structural element of any format shown in FIG. 1.

[0096] In some embodiments, the provided single-stranded RNAi agents have any format shown in FIG. 1, or any structural element of any format shown in FIG. 1.

[0097] The data provided by the present disclosure in particular show that various oligonucleotides of the disclosed formats are capable of directing the reduction of the expression and / or level of a target gene or its gene product in any one of a number of different genes (when targeted against any one of a number of different sequences). In some embodiments, the data provided by the present disclosure show that various RNAi agents of the disclosed formats are capable of directing RNA interference against any one of a number of different sequences in any one of a number of different genes.

[0098] In some embodiments, an APOC3 oligonucleotide having any of the structures described and / or shown herein is capable of directing RNA interference. In some embodiments, an APOC3 oligonucleotide having any of the structures described and / or shown herein is capable of directing RNase H-mediated knockdown. In some embodiments, an APOC3 oligonucleotide having any of the structures described and / or shown herein is capable of directing RNA interference and / or RNase H-mediated knockdown. In some embodiments, the APOC3 oligonucleotide comprises any oligonucleotide described herein or any structural element in any format described herein or shown in FIG. 1. In some embodiments, the APOC3 oligonucleotide comprises any oligonucleotide described herein or any structural element in any format described herein or shown in FIG. 1 and is capable of directing RNA interference. In some embodiments, the APOC3 oligonucleotide comprises any oligonucleotide described herein or any structural element in any format described herein or shown in FIG. 1 and is capable of directing RNase H-mediated knockdown. In some embodiments, the APOC3 oligonucleotide comprises any oligonucleotide described herein or any structural element in any format described herein or shown in FIG. 1 and is capable of directing RNA interference and / or RNase H-mediated knockdown.

[0099] In some embodiments, the RNAi agent comprises one or more of the following: a 5′-terminal structure, a 5′-terminal region, a seed region, a post-seed region, and a 3′-terminal region and optionally additional chemical moieties. In some embodiments, the seed region is any seed region described herein or known in the art. In some embodiments, the post-seed region can be any region between a seed region described herein or known in the art and the 3′-terminal region. In some embodiments, the 3′-terminal region can be any 3′-terminal region described herein or known in the art. In some embodiments, any optionally additional chemical moiety can be any optionally additional chemical moiety described herein or known in the art. Any individual 5′-terminal structure, 5′-terminal region, seed region, post-seed region, 3′-terminal region, and optionally additional chemical moiety described herein or known in the art can be combined independently with any other 5′-terminal structure, 5′-terminal region, seed region, post-seed region, 3′-terminal region, and optionally additional chemical moiety described herein or known in the art. In some embodiments, by way of non-limiting example, the regions of a single-stranded RNAi agent are a 5′-terminal structure, a 5′-terminal region, a seed region, a post-seed region, a portion of the seed region, a portion of the post-seed region, or a 3′-terminal dinucleotide.

[0100] In some embodiments, the provided oligonucleotide has a base sequence consisting of the base sequence of any oligonucleotide disclosed herein. In some embodiments, the provided oligonucleotide has a base sequence comprising the base sequence of any oligonucleotide disclosed herein. In some embodiments, the provided oligonucleotide has a base sequence comprising the following sequence which comprises a sequence of 15 consecutive bases of the base sequence of any oligonucleotide disclosed herein. In some embodiments, the provided oligonucleotide has a base sequence comprising the following sequence which comprises a sequence of 20 consecutive bases (with at most 5 mismatches) of the base sequence of any oligonucleotide disclosed herein.

[0101] In some embodiments, the provided oligonucleotide is capable of directing a decrease in the expression and / or level of a target gene or its gene product. In some embodiments, the provided oligonucleotide is capable of directing single-stranded RNAi interference. In some embodiments, the provided oligonucleotide is capable of directing RNase H-mediated knockdown. In some embodiments, the provided oligonucleotide is capable of directing both single-stranded RNA interference and RNase H-mediated knockdown. In some embodiments, the oligonucleotide comprises a sequence that targets any transcript or gene targeted by the oligonucleotides disclosed herein.

[0102] In some embodiments, the provided oligonucleotide targets APOC3.

[0103] In some embodiments, the provided oligonucleotide can be used to reduce or inhibit the activity, level, and / or expression of the APOC3 gene or its gene product. In some embodiments, the provided oligonucleotide can be used to reduce or inhibit the activity, level, and / or expression of a gene or its gene product, wherein the abnormal or excessive activity, level, and / or expression of the gene or its gene product, a harmful mutation in the gene or its gene product, or the tissue or intercellular or intracellular distribution of the gene or its gene product is associated with, causes, and / or is linked to a disorder. In some embodiments, the provided oligonucleotide can be used to treat a disorder and / or prepare a medicament for treating a disorder, the disorder being associated with, caused by, and / or linked to the abnormal or excessive activity, level, and / or expression or abnormal distribution of a gene or its gene product.

[0104] In some embodiments, the present disclosure relates to methods of using the oligonucleotides disclosed herein that are capable of targeting APOC3 and can be used to treat APOC3-related disorders and / or prepare treatments for APOC3-related disorders.

[0105] In some embodiments, an APOC3 oligonucleotide capable of targeting a gene comprises a base sequence that is part of the base sequence of the target gene or is complementary or substantially complementary to a part of the base sequence of the target gene. In some embodiments, the length of the part is at least 15 bases. In some embodiments, the base sequence of a single-stranded RNAi agent can comprise or consist of a base sequence that has a specified maximum number of mismatches with a specified base sequence.

[0106] In some embodiments, a mismatch is a difference between base sequences or lengths when two sequences are maximally aligned and compared. As a non-limiting example, if there is a difference between the base at a particular position in one sequence and the base at the corresponding position in the other sequence, it counts as a mismatch. Thus, for example, if a position in one sequence has a particular base (e.g., A) and the corresponding position in the other sequence has a different base (e.g., G, C, or U), it counts as a mismatch. For example, if a position in one sequence has a base (e.g., A) and the corresponding position in the other sequence has no base (e.g., the position is a abasic nucleotide that contains a phosphosugar backbone but no base) or skips the position, it also counts as a mismatch. A single-stranded nick in either sequence (or the sense or antisense strand) may not count as a mismatch. For example, if one sequence contains the sequence 5′-AG-3′ and the other sequence contains the sequence 5′-AG-3′ with a single-stranded nick between A and G, it will not count as a mismatch. Base modifications are generally not considered mismatches. For example, if one sequence contains C and the other sequence contains a modified C (e.g., 5mC) at the same position, it may not count as a mismatch. In some embodiments, for the purpose of counting mismatches, substituting T for U or vice versa is not considered a mismatch.

[0107] In some embodiments, the APOC3 oligonucleotide is complementary or fully or 100% complementary to a target sequence (e.g., RNA, such as mRNA), meaning that there are no mismatches between the base sequence of the oligonucleotide and a sequence that is fully complementary to the target gene (e.g., base pairs by Watson-Crick base pairing). Without being bound by any particular theory, the present disclosure states that for single-stranded RNAi agents, the 5'-terminal nucleotide portion or the 3'-terminal dinucleotide need not base pair with the target. These can be mismatches. Additionally, an antisense oligonucleotide or single-stranded RNAi agent may have a small number of internal mismatches and still direct a reduction in the expression and / or level of the target gene or its gene product, and / or direct RNase H-mediated knockdown and / or RNA interference. If the first base sequence of the APOC3 oligonucleotide (e.g., antisense oligonucleotide or single-stranded RNAi agent) has a small number of mismatches with a reference base sequence that is 100% complementary to the target sequence, then the first base sequence is substantially complementary to the target sequence. In some embodiments, the APOC3 oligonucleotide (e.g., antisense oligonucleotide or single-stranded RNAi agent) can have a base sequence that is complementary or substantially complementary to the target sequence. In some embodiments, complementarity is determined based on Watson-Crick base pairing (guanine-cytosine and adenine-thymine / uracil), where guanine, cytosine, adenine, thymine, uracil can be optionally and independently modified but retain their pairing hydrogen bond patterns when unmodified. In some embodiments, a sequence that is complementary to another sequence contains at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 bases.

[0108] In some embodiments, the APOC3 oligonucleotide, oligonucleotide composition, or oligonucleotide type has a common backbone linkage pattern. In some embodiments, the common backbone linkage pattern contains at least 10 modified internucleotide linkages.

[0109] In some embodiments, the common backbone linkage pattern contains at least 10 phosphorothioate linkages. In some embodiments, the APOC3 oligonucleotide, oligonucleotide composition, or oligonucleotide type has a common backbone chiral center pattern. In some embodiments, the common backbone chiral center pattern contains at least 1 internucleotide linkage of the Sp configuration. In some embodiments, the common backbone chiral center pattern contains at least 1 internucleotide linkage that is a phosphorothioate of the Sp configuration. In some embodiments, the oligonucleotides in the provided composition have a common backbone phosphorus modification pattern. In some embodiments, the provided composition is a chirally controlled APOC3 oligonucleotide composition because the composition contains a predetermined level of oligonucleotides of a single oligonucleotide type, where the APOC3 oligonucleotide type is defined by:

[0110] 1) Base sequence;

[0111] 2) Backbone linkage pattern;

[0112] 3) Backbone chiral center pattern; and

[0113] 4) Backbone phosphorus modification pattern.

[0114] As noted above and as understood in the art, in some embodiments, the base sequence of an APOC3 oligonucleotide can refer to the identity and / or modification status of the nucleoside residues in the oligonucleotide (e.g., the sugar and / or base moieties, relative to standard naturally occurring nucleotides such as adenine, cytosine, guanosine, thymine, and uracil) and / or the hybridization characteristics of such residues (i.e., the ability to hybridize to specific complementary residues).

[0115] In some embodiments, a particular type of oligonucleotide can be defined by:

[0116] 1A) Base identity;

[0117] 1B) Base modification pattern;

[0118] 1C) Sugar modification pattern;

[0119] 2) Backbone linkage pattern;

[0120] 3) Backbone chiral center pattern; and

[0121] 4) Backbone phosphorus modification pattern.

[0122] Thus, in some embodiments, a particular type of oligonucleotide can share the same bases, but have different patterns of base modification and / or sugar modification. In some embodiments, a particular type of oligonucleotide can share the same bases and pattern of base modification (including, for example, the absence of base modification), but have different patterns of sugar modification.

[0123] In some embodiments, a particular type of oligonucleotide is chemically identical because it has the same base sequence (including length), the same pattern of chemical modification of the sugar and base moieties, the same backbone linkage pattern (e.g., the pattern of natural phosphate linkages, phosphorothioate linkages, phosphorothioate triesters, and combinations thereof), the same backbone chiral center pattern (e.g., the stereochemical pattern of chiral internucleotide linkages (Rp / Sp)), and the same backbone phosphorus modification pattern (e.g., the pattern of modification of the phosphorus atoms between nucleotides, such as -S - and -L-R of formula I 1 ).

[0124] The present disclosure particularly provides oligonucleotide compositions and techniques for optimizing properties (e.g., improved single-stranded RNA interference, RNase H-mediated knockdown, etc.). In some embodiments, the present disclosure provides methods for reducing the immune response associated with the administration of oligonucleotides and their compositions (i.e., methods of administering an oligonucleotide composition such that an adverse immune response to the oligonucleotides in the composition is reduced, e.g., relative to an adverse immune response observed with a reference composition of nucleotides having a comparable or identical nucleotide sequence). In some embodiments, the present disclosure provides methods for increasing the binding to certain proteins by oligonucleotides and their compositions. In some embodiments, the present disclosure provides methods for increasing the binding to certain proteins by oligonucleotides and their compositions. In some embodiments, the present disclosure provides methods for enhancing the delivery of oligonucleotides and their compositions. The present disclosure particularly encompasses the recognition that, in some embodiments, optimal delivery of an oligonucleotide to its target involves a balance of oligonucleotide binding to certain proteins such that the oligonucleotide can be transported to the desired location and involves release of the oligonucleotide such that the oligonucleotide can be appropriately released from certain proteins to perform its desired function, e.g., hybridize to its target, cleave its target, inhibit translation, regulate transcript processing, etc. As exemplified in the present disclosure, the present disclosure particularly recognizes that improvements in oligonucleotide properties can be achieved through chemical modification and / or stereochemistry.

[0125] In some embodiments, the present disclosure provides a method for treating or preventing a disease, the method comprising administering to a subject an APOC3 oligonucleotide composition described herein.

[0126] In some embodiments, the disease is a disease in which knockdown of a target nucleic acid by single-stranded RNA interference can repair, restore, or introduce a new beneficial function after administration of the provided composition.

[0127] In some embodiments, the common sequence comprises a sequence selected from Table 1A. In some embodiments, the common sequence is a sequence selected from Table 1A. In some embodiments, the backbone chiral center pattern is selected from those described in Table 1A.

[0128] In some embodiments, the present disclosure provides a method that comprises administering a composition comprising a first plurality of oligonucleotides, the composition exhibiting improved delivery compared to a reference composition comprising a plurality of oligonucleotides, each of the plurality of nucleotides also having a common base sequence but being structurally different from the first plurality of oligonucleotides in that:

[0129] each of the oligonucleotides in the reference plurality of oligonucleotides is structurally different from one another in stereochemistry; and / or

[0130] At least some of the oligonucleotides in the reference plurality of oligonucleotides have a structure that is different from the structure represented by the plurality of oligonucleotides of the composition.

[0131] In some embodiments, the present disclosure provides a method of administering an APOC3 oligonucleotide composition comprising a first plurality of oligonucleotides capable of directing a reduction in expression and / or levels of a target gene or its gene product and having a common nucleotide sequence, wherein the improvement comprises:

[0132] Administration of an APOC3 oligonucleotide composition comprising a first plurality of oligonucleotides is characterized by improved delivery relative to a reference oligonucleotide composition having the same common nucleotide sequence.

[0133] In some embodiments, the present disclosure provides a method of administering an APOC3 oligonucleotide composition comprising a first plurality of oligonucleotides capable of directing single-stranded RNA interference and having a common nucleotide sequence, wherein the improvement comprises:

[0134] Administration of an APOC3 oligonucleotide composition comprising a first plurality of oligonucleotides is characterized by improved delivery relative to a reference oligonucleotide composition having the same common nucleotide sequence.

[0135] In some embodiments, the present disclosure provides a single-stranded RNAi agent selected from any table disclosed herein (including but not limited to Table 1A) or elsewhere APOC3 oligonucleotides. In some embodiments, the present disclosure provides a single-stranded RNAi agent selected from any table disclosed herein (including but not limited to Table 1A) or elsewhere APOC3 oligonucleotides, wherein the oligonucleotide is conjugated to a lipid portion.

[0136] In some embodiments, the oligonucleotide provided comprises a lipid portion. In some embodiments, the lipid portion is incorporated by conjugation with a lipid. In some embodiments, the lipid portion is a fatty acid. In some embodiments, the APOC3 oligonucleotide is conjugated with a fatty acid. In some embodiments, the single-stranded RNAi agent provided further comprises a lipid. In some embodiments, the single-stranded RNAi agent provided comprises a lipid portion conjugated at the 9th or 11th nucleotide (counting from the 5'-end). In some embodiments, the APOC3 oligonucleotide is conjugated with a fatty acid at the base. In some embodiments, the single-stranded RNAi agent provided comprises a lipid portion. In some embodiments, the single-stranded RNAi agent provided comprises a lipid portion conjugated at the base at the 9th or 11th nucleotide (counting from the 5'-end).

[0137] In some embodiments, the single-stranded RNAi agent is any of the aforementioned compositions, further comprising one or more additional components.

[0138] In some embodiments, the provided oligonucleotides are capable of degrading target transcripts, such as RNA, via the RNase H mechanism and the RNAi mechanism.

[0139] In some embodiments, the conjugation of the lipid moiety to the APOC3 oligonucleotide improves at least one property of the oligonucleotide. In some embodiments, the improved properties include increased activity (e.g., increased ability to direct the expression and / or reduction of the level of a target gene or its gene product, and / or to direct single-stranded RNA interference, and / or to direct RNase H-mediated knockdown) and / or improved tissue distribution. In some embodiments, the tissue is muscle tissue. In some embodiments, the tissue is skeletal muscle, gastrocnemius, triceps, heart, or diaphragm. In some embodiments, the improved property includes reduced hTLR9 agonist activity. In some embodiments, the improved property includes hTLR9 antagonist activity. In some embodiments, the improved property includes increased hTLR9 antagonist activity.

[0140] Generally, the properties of the oligonucleotide compositions described herein can be evaluated using any suitable assay.

[0141] Those skilled in the art will recognize and / or will be able to readily develop suitable assays for specific oligonucleotide compositions.

[0142] Definitions

[0143] As used herein, unless otherwise specified, the following definitions apply. For the purposes of this disclosure, chemical elements are identified according to the Periodic Table of the Elements (CAS version) and Handbook of Chemistry and Physics, 75th Edition. In addition, general principles of organic chemistry are described in "Organic Chemistry", Thomas Sorrell, University Science Books, Sausalito: 1999, and "March's Advanced Organic Chemistry", 5th Edition, Smith, M.B. and March, J., eds., John Wiley & Sons, New York: 2001.

[0144] As used herein in the specification, "a" or "an" can mean one or more. As used herein in the claims, when used in conjunction with the word "comprising", the words "a" or "an" can mean one or more than one. As used herein, "another" can refer to at least a second or more.

[0145] The term "about" means an approximation of plus or minus 10% of the nominal value to which it refers, or in one embodiment, plus or minus 5%, or in another embodiment, plus or minus 2%. For the field of the present disclosure, this level of approximation is appropriate unless a more stringent range is required for the stated value.

[0146] Aliphatic: As used herein, "aliphatic" means a straight-chain (i.e., unbranched) or branched substituted or unsubstituted hydrocarbon chain that is fully saturated or contains one or more unsaturated units, or a substituted or unsubstituted monocyclic, bicyclic or polycyclic hydrocarbon ring that is fully saturated or contains one or more unsaturated units (but does not include aromatic units), or a combination thereof. In some embodiments, the aliphatic group contains 1-50 aliphatic carbon atoms. In some embodiments, the aliphatic group contains 1-20 aliphatic carbon atoms. In other embodiments, the aliphatic group contains 1-10 aliphatic carbon atoms. In other embodiments, the aliphatic group contains 1-9 aliphatic carbon atoms. In other embodiments, the aliphatic group contains 1-8 aliphatic carbon atoms. In other embodiments, the aliphatic group contains 1-7 aliphatic carbon atoms. In other embodiments, the aliphatic group contains 1-6 aliphatic carbon atoms. In other embodiments, the aliphatic group contains 1-5 aliphatic carbon atoms, and in still other embodiments, the aliphatic group contains 1, 2, 3 or 4 aliphatic carbon atoms. Suitable aliphatic groups include, but are not limited to, straight-chain or branched substituted or unsubstituted alkyl, alkenyl, alkynyl, and hybrids thereof, such as (cycloalkyl)alkyl, (cycloalkenyl)alkyl or (cycloalkyl)alkenyl.

[0147] Alkenyl: As used herein, the term "alkenyl" refers to an alkyl as defined herein having one or more double bonds.

[0148] Alkyl: As used herein, the term "alkyl" has its ordinary meaning in the art and can include saturated aliphatic groups, including straight-chain alkyls, branched-chain alkyls, cycloalkyl (alicyclic) groups, alkyl-substituted cycloalkyls and cycloalkyl-substituted alkyls. In some embodiments, the alkyl has 1-100 carbon atoms. In certain embodiments, the straight-chain or branched-chain alkyl has about 1-20 carbon atoms in its backbone (e.g., C1-C 20 , for a straight chain, and C2-C 20), and optionally having from about 1 to 10 carbon atoms. In some embodiments, the cycloalkyl ring has from about 3 to 10 carbon atoms in its ring structure, such rings being monocyclic, bicyclic or polycyclic, and optionally having about 5, 6 or 7 carbons in said ring structure. In some embodiments, the alkyl can be a lower alkyl, where lower alkyl contains 1-4 carbon atoms (e.g., C1-C4 for straight-chain lower alkyl).

[0149] Alkynyl: As used herein, the term “alkynyl” refers to an alkyl as defined herein having one or more triple bonds.

[0150] Antisense: As used herein, the term “antisense” refers to an oligonucleotide or other nucleic acid having a base sequence that is complementary or substantially complementary to a target nucleic acid capable of hybridizing thereto. In some embodiments, the target nucleic acid is target gene mRNA. In some embodiments, hybridization is required for or results in an activity, e.g., a decrease in the level, expression or activity of the target nucleic acid or its gene product. As used herein, the term “translating oligonucleotide” refers to an oligonucleotide complementary to a target nucleic acid. In some embodiments, an antisense oligonucleotide is capable of directing a decrease in the level, expression or activity of a target nucleic acid or its gene product. In some embodiments, an antisense oligonucleotide is capable of directing a decrease in the level, expression or activity of a target nucleic acid or its gene product through a mechanism involving RNase H, steric hindrance and / or RNA interference.

[0151] About: As used herein, unless otherwise stated or otherwise apparent from the context, the term “about” or “approximately” with respect to a numerical value generally is considered to include numerical values that fall within the range of 5%, 10%, 15% or 20% in either direction (greater than or less than) of the stated value (except in instances where such values would be less than 0% of the possible value or exceed 100% of the possible value). In some embodiments, use of the term “about” with respect to a dose means ±5 mg / kg / day.

[0152] Aryl: As used herein, the term "aryl", used alone or as part of a larger moiety such as "aralkyl", "aralkoxy", or "aryloxyalkyl", refers to a monocyclic, bicyclic, or polycyclic ring system having a total of five to thirty ring members, wherein at least one of the rings in the system is aromatic. In some embodiments, aryl is a monocyclic, bicyclic, or polycyclic ring system having a total of five to fourteen ring members, wherein at least one of the rings in the system is aromatic and wherein each ring in the system contains 3 to 7 ring members. In some embodiments, aryl is a biaryl. The term "aryl" is used interchangeably with the term "aryl ring". In certain embodiments of the present disclosure, "aryl" refers to an aromatic ring system that may carry one or more substituents, including but not limited to phenyl, biphenyl, naphthyl, binaphthyl, anthryl, and the like. Also included within the scope of the term "aryl" as used herein are groups in which an aromatic ring is fused to one or more non-aromatic rings, such as indanyl, phthalimido, naphthalimido, phenanthridinyl, or tetrahydronaphthyl, and the like.

[0153] Characteristic portion: As used herein, the phrase "characteristic portion" of a protein or polypeptide is the portion that contains a collection of contiguous amino acids or multiple collections of contiguous amino acids that together serve as a characteristic of the protein or polypeptide. Each such contiguous segment will generally contain at least two amino acids. Generally, a characteristic portion is a portion that, in addition to the sequence identity specified above, also shares at least one functional characteristic with the relevant full-length protein.

[0154] Characteristic structural element: The term "characteristic structural element" or "structural element" refers to a unique structural element that is present in all members of a family of polypeptides, small molecules, or nucleic acids and can thus be used by one of ordinary skill in the art to define the members of the family. In some embodiments, the structural elements of a single-stranded RNAi agent include, but are not limited to: 5'-terminal structure, 5'-terminal region, 5'-nucleotide portion, seed region, post-seed region, 3'-terminal region, 3'-terminal dinucleotide, 3'-cap, modification pattern, stereochemical pattern in the backbone, additional chemical moieties, and the like.

[0155] Comparable: The term "comparable" is used herein to describe two (or more) sets of conditions or circumstances that are sufficiently similar to each other to allow comparison of the results obtained or the phenomena observed. In some embodiments, multiple sets of comparable conditions or circumstances are characterized by a plurality of substantially identical characteristics and one or a few different characteristics. One of ordinary skill in the art will understand that when multiple sets of conditions are characterized by a sufficient number and type of substantially identical characteristics, the multiple sets of conditions are comparable to each other to warrant the reasonable conclusion that differences in the results obtained or the phenomena observed under different sets of conditions or circumstances are caused by or indicative of changes in those different characteristics.

[0156] Cycloaliphatic: The terms "cycloaliphatic", "carbocycle", "carbocyclyl", "carbocyclic radical", and "carbocyclic ring" are used interchangeably and, as used herein, refer to a saturated or partially unsaturated but non-aromatic cyclic aliphatic monocyclic, bicyclic, or polycyclic ring system having 3 to 30 ring members as described herein, unless otherwise specified. Cycloaliphatic groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cycloheptyl, cycloheptenyl, cyclooctyl, cyclooctenyl, norbornyl, adamantyl, and cyclooctadienyl. In some embodiments, the cycloaliphatic group has 3-6 carbons. In some embodiments, the cycloaliphatic group is saturated and is a cycloalkyl. The term "cycloaliphatic" can also include an aliphatic ring fused to one or more aromatic or non-aromatic rings (such as decahydronaphthyl or tetrahydronaphthyl). In some embodiments, the cycloaliphatic group is bicyclic. In some embodiments, the cycloaliphatic group is tricyclic. In some embodiments, the cycloaliphatic group is polycyclic. In some embodiments, "cycloaliphatic" refers to a C3-C6 monocyclic hydrocarbon, or a C8-C 10 bicyclic or polycyclic hydrocarbon that is fully saturated or contains one or more unsaturated but non-aromatic units and has a single point of attachment to the remainder of the molecule, or a C9-C 16 polycyclic hydrocarbon that is fully saturated or contains one or more unsaturated but non-aromatic units and has a single point of attachment to the remainder of the molecule.

[0157] Heteroaliphatic: As used herein, the term "heteroaliphatic" has its ordinary meaning in the art and refers to an aliphatic group in which one or more carbon atoms are independently replaced by one or more heteroatoms (e.g., oxygen, nitrogen, sulfur, silicon, phosphorus, etc.). In some embodiments, one or more units selected from C, CH, CH2, and CH3 are independently replaced by one or more heteroatoms (including their oxidized and / or substituted forms). In some embodiments, the heteroaliphatic group is a heteroalkyl. In some embodiments, the heteroaliphatic group is a heteroalkenyl.

[0158] Heteroalkyl: As used herein, the term "heteroalkyl" has its ordinary meaning in the art and refers to an alkyl in which one or more carbon atoms are independently replaced by one or more heteroatoms (e.g., oxygen, nitrogen, sulfur, silicon, phosphorus, etc.). Examples of heteroalkyls include, but are not limited to, alkoxy, poly(ethylene glycol)-, alkyl-substituted amino, tetrahydrofuranyl, piperidinyl, morpholinyl, etc.

[0159] Heteroaryl: As used herein, the terms "heteroaryl" and "heteroar-" when used alone or as part of a larger moiety (such as "heteroalkyl" or "heteroalkoxy") refer to a monocyclic, bicyclic or polycyclic ring system having a total of five to thirty ring members, wherein at least one ring in the system is aromatic and at least one aromatic ring atom is a heteroatom. In some embodiments, heteroaryl is a group having 5 to 10 ring atoms (i.e., monocyclic, bicyclic or polycyclic), and in some embodiments, is a group having 5, 6, 9 or 10 ring atoms. In some embodiments, heteroaryl has 6, 10 or 14 π electrons shared in a cyclic array; and in addition to carbon atoms, has one to five heteroatoms. Heteroaryl includes, but is not limited to, thienyl, furyl, pyrrolyl, imidazolyl, pyrazolyl, triazolyl, tetrazolyl, oxazolyl, isoxazolyl, oxadiazolyl, thiazolyl, isothiazolyl, thiadiazolyl, pyridyl, pyridazinyl, pyrimidinyl, pyrazinyl, indolizinyl, purinyl, naphthyridinyl and pteridinyl. In some embodiments, heteroaryl is heteroaryl, such as bipyridyl and the like. As used herein, the terms "heteroaryl" and "heteroar-" also include groups in which a heteroaromatic ring is fused to one or more aryl, cycloaliphatic or heterocyclic rings, wherein the linking group or point of attachment is on the heteroaromatic ring. Non-limiting examples include indolyl, isoindolyl, benzothienyl, benzofuryl, dibenzofuryl, indazolyl, benzimidazolyl, benzothiazolyl, quinolinyl, isoquinolinyl, cinnolinyl, phthalazinyl, quinazolinyl, quinoxalinyl, 4H-quinolizinyl, carbazolyl, acridinyl, phenazinyl, phenothiazinyl, phenoxazinyl, tetrahydroquinolinyl, tetrahydroisoquinolinyl and pyrido[2,3-b]-1,4-oxazin-3(4H)-one. Heteroaryl can be monocyclic, bicyclic or polycyclic. The term "heteroaryl" can be used interchangeably with the terms "heteroaryl ring", "heteroaryl group" or "heteroaromatic", any of which includes an optionally substituted ring. The term "heteroalkyl" refers to an alkyl group substituted by a heteroaryl group, wherein the alkyl and heteroaryl moieties are independently optionally substituted.

[0160] Heteroatom: As used herein, the term "heteroatom" means an atom that is not carbon or hydrogen. In some embodiments, the heteroatom is oxygen, sulfur, nitrogen, phosphorus or silicon (including any oxidized form of nitrogen, sulfur, phosphorus or silicon; any quaternized form of a basic nitrogen; or a replaceable nitrogen of a heterocycle (e.g., N in 3,4-dihydro-2H-pyrrolyl), NH (as in pyrrolidinyl) or NR + (as in N-substituted pyrrolidinyl), etc.).

[0161] Heterocycle: As used herein, the terms "heterocycle", "heterocyclyl", "heterocyclic radical", and "heterocyclic ring" are used interchangeably and refer to a saturated or partially unsaturated monocyclic, bicyclic, or polycyclic moiety (e.g., 3 - 30 membered) having one or more heteroatom ring atoms. In some embodiments, the heterocyclyl is a stable 5 - to 7 - membered monocyclic or 7 - to 10 - membered bicyclic heterocyclic moiety, which is saturated or partially unsaturated and, in addition to carbon atoms, has one or more, preferably one to four, heteroatoms as defined above. When referring to the ring atoms of a heterocycle, the term "nitrogen" includes substituted nitrogen. For example, in a saturated or partially unsaturated ring having 0 - 3 heteroatoms selected from oxygen, sulfur, and nitrogen, nitrogen can be N (as in 3,4 - dihydro - 2H - pyrrolyl), NH (as in pyrrolidinyl), or + NR (as in N - substituted pyrrolidinyl). The heterocycle can be attached to its side group at any heteroatom or carbon atom that results in a stable structure, and any ring atom can be optionally substituted. Examples of such saturated or partially unsaturated heterocyclyls include, but are not limited to, tetrahydrofuranyl, tetrahydrothienyl, pyrrolidinyl, piperidinyl, pyrrolinyl, tetrahydroquinolinyl, tetrahydroisoquinolinyl, decahydroquinolinyl, oxazolidinyl, piperazinyl, dioxolanyl, dioxolanyl, diazepinyl, oxazepinyl, thiazepinyl, morpholinyl, and quinuclidinyl. The terms "heterocycle", "heterocyclyl", "heterocyclyl ring", "heterocyclic group", "heterocyclic moiety", and "heterocyclic radical" are used interchangeably herein and also include groups in which a heterocyclyl ring is fused to one or more aryl, heteroaryl, or cycloaliphatic rings, such as indolinyl, 3H - indolyl, chromanyl, phenanthridinyl, or tetrahydroquinolinyl. The heterocyclyl can be monocyclic, bicyclic, or polycyclic. The term "heterocyclylalkyl" refers to an alkyl group substituted with a heterocyclyl, wherein the alkyl and heterocyclyl moieties are independently optionally substituted.

[0162] Lower alkyl: The term "lower alkyl" refers to a C 1-4 straight or branched chain alkyl. Exemplary lower alkyls are methyl, ethyl, propyl, isopropyl, butyl, isobutyl, and tert - butyl.

[0163] Lower haloalkyl: The term "lower haloalkyl" refers to a C 1-4 straight or branched chain alkyl substituted with one or more halo atoms.

[0164] Optionally substituted: As described herein, the compounds of the present disclosure (e.g., oligonucleotides) may contain optionally substituted and / or substituted moieties. In general, the term "substituted", whether or not preceded by the term "optionally", means that one or more hydrogens of the designated moiety are replaced by a suitable substituent. Unless otherwise specified, an "optionally substituted" group may have a suitable substituent at each substitutable position of the group, and when more than one position in any given structure is available for substitution by more than one substituent selected from the designated group, the substituents may be the same or different at each position. In some embodiments, the optionally substituted group is unsubstituted. Combinations of substituents contemplated by the present disclosure are preferably those that result in the formation of stable or chemically viable compounds. As used herein, the term "stable" refers to a compound that does not substantially change when subjected to conditions that permit their production, detection, and in some embodiments, their recovery, purification, and use for one or more of the purposes disclosed herein.

[0165] Suitable monovalent substituents on a substitutable atom (e.g., a suitable carbon atom) are independently halogen; -(CH2) 0-4 R o ; -(CH2) 0-4 OR o ; -O(CH2) 0-4 R o ; -O-(CH2) 0-4 C(O)OR o ; -(CH2) 0-4 CH(OR o )2; substituted by R o -(CH2) 0-4 Ph; substituted by R o -(CH2) 0-4 O(CH2) 0-1 Ph; substituted by R o -CH=CHPh; may be substituted by R o -(CH2) 0-4 O(CH2) 0-1 -pyridyl; -NO2 -CN; -N3; -(CH2) 0-4 N(R o )2; -(CH2) 0-4 N(R o )C(O)R o ; -N(R o )C(S)R o ; -(CH2) 0-4 N(R o )C(O)NR o 2; -N(R o )C(S)NRo 2; -(CH2) 0-4 N(R o )C(O)OR o ; -N(R o )N(R o )C(O)R o ; -N(R o )N(R o )C(O)NR o 2; -N(R o )N(R o )C(O)OR o ; -(CH2) 0-4 C(O)R o ; -C(S)R o ; -(CH2) 0-4 C(O)OR o ; -(CH2) 0-4 C(O)SR o ; -(CH2) 0-4 C(O)OSiR o 3; -(CH2) 0-4 OC(O)R o ; -OC(O)(CH2) 0-4 SR; -SC(S)SR o ; -(CH2) 0-4 SC(O)R o ; -(CH2) 0-4 C(O)NR o 2; -C(S)NR o 2; -C(S)SR o ; -SC(S)SR o ; -(CH2) 0-4 OC(O)NR o 2; -C(O)N(OR o )R o ; -C(O)C(O)R o ; -C(O)CH2C(O)R o ; -C(NOR o )R o ; -(CH2) 0-4 SSR o ; -(CH2) 0-4 S(O)2R o ; -(CH2) 0-4 S(O)2OR o ; -(CH2) 0-4 OS(O)2R o ; -S(O)2NR o 2; -(CH2)0-4 S(O)R o ; -N(R o )S(O)2NR o 2; -N(R o )S(O)2R o ; -N(OR o )R o ; -C(NH)NR o 2; -Si(R o )3; -O Si(R o )3; -B(R o )2; -OB(R o )2; -OB(OR o )2; -P(R o )2; -P(OR o )2; -OP(R o )2; -OP(OR o )2; -P(O)(R o )2; -P(O)(OR o )2; -OP(O)(R o )2; -OP(O)(OR o )2; -OP(O)(OR o )(SR o ); -SP(O)(R o )2; -SP(O)(OR o )2; -N(R o )P(O)(R o )2; -N(R o )P(O)(OR o )2; -P(R o )2[B(R o )3]; -P(OR o )2[B(R o )3]; -OP(R o )2[B(R o )3]; -OP(OR o )2[B(R o )3]; -(C 1-4 linear or branched alkylene)O-N(R o )2; or -(C 1-4 linear or branched alkylene)C(O)O-N(R o )2, where each R o can be substituted as defined below and is independently hydrogen, C 1-20 aliphatic, C with 1 - 5 heteroatoms independently selected from nitrogen, oxygen, sulfur, silicon, and phosphorus 1-20 heteroaliphatic, -CH2-(C 6-14(aryl)-O(CH2) 0-1 (C 6-14 (aryl)-CH2-(5- to 14-membered heteroaryl ring), a 5- to 20-membered monocyclic, bicyclic or polycyclic ring having 0 to 5 heteroatoms independently selected from nitrogen, oxygen, sulfur, silicon and phosphorus, a saturated, partially unsaturated or aryl ring, or notwithstanding the above definition, two independently occurring R o and the intervening atoms together form a 5- to 20-membered monocyclic, bicyclic or polycyclic, saturated, partially unsaturated or aryl ring having 0 to 5 heteroatoms independently selected from nitrogen, oxygen, sulfur, silicon and phosphorus, which may be substituted as defined below.

[0166] R o (or the ring formed by two independently occurring R o and the intervening atoms together) suitable monovalent substituents are independently halogen, -(CH2) 0-2 R · , -(halo-R · ), -(CH2) 0-2 OH, -(CH2) 0-2 OR · , -(CH2) 0-2 CH(OR · )2, -O(halo-R · ), -CN, -N3, -(CH2) 0-2 C(O)R · , -(CH2) 0-2 C(O)OH, -(CH2) 0-2 C(O)OR · , -(CH2) 0-2 SR · , -(CH2) 0- 2SH, -(CH2) 0-2 NH2, -(CH2) 0-2 NHR · , -(CH2) 0-2 NR · 2, -NO2, -SiR · 3, -OSiR · 3, -C(O)SR · , -(C 1-4 linear or branched alkylene)C(O)OR · or -SSR · , where each R · is unsubstituted or when preceded by "halo" is substituted by only one or more halogens and is independently selected from C 1-4 aliphatic, -CH2Ph, -O(CH2) 0-1Ph, and a 5-6 membered saturated, partially unsaturated or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen and sulfur. R o Suitable divalent substituents on the saturated carbon atoms of R include =O and =S.

[0167] For example, suitable divalent substituents on suitable carbon atoms are independently the following: =O, =S, =NNR * 2, =NNHC(O)R * , =NNHC(O)OR * , =NNHS(O)2R * , =NR * , =NOR * , -O(C(R * 2)) 2-3 O- or -S(C(R * 2)) 2-3 S-, where each independently occurring R * is selected from hydrogen, C 1-6 aliphatic which may be substituted as defined hereinafter, and an unsubstituted 5-6 membered saturated, partially unsaturated or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen and sulfur. Suitable divalent substituents bonded to the ortho-substitutable carbon of an "optionally substituted" group include: -O(CR * 2) 2-3 O-, where each independently occurring R * is selected from hydrogen, C1-6 aliphatic which may be substituted as defined hereinafter, and an unsubstituted 5-6 membered saturated, partially unsaturated or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen and sulfur.

[0168] R * Suitable substituents on the aliphatic group of are independently halogen, -R · , -(halo-R · ), -OH, -OR · , -O(halo-R · ), -CN, -C(O)OH, -C(O)OR · , -NH2, -NHR · , -NR · 2 or -NO2, where each R · is unsubstituted or, when preceded by "halo", is substituted by one or more halogens only, and is independently C 1-4 aliphatic, -CH2Ph, -O(CH2) 0-1 Ph, or a 5-6 membered saturated, partially unsaturated and or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen and sulfur.

[0169] Partially unsaturated: As used herein, the term "partially unsaturated" refers to a cyclic moiety that includes at least one double or triple bond. The term "partially unsaturated" is intended to encompass rings having multiple sites of unsaturation and is not intended to include aryl or heteroaryl moieties as defined herein.

[0170] RNA interference: As used herein, the term "RNA interference" or "RNAi" refers to a post-transcriptional targeted gene silencing process involving RISC (RNA-induced silencing complex). It has been reported that the process of RNAi occurs naturally when the ribonuclease III (Dicer) cleaves longer dsRNA into shorter fragments (referred to as siRNA). Naturally occurring siRNAs (small interfering RNAs) are typically about 21 to 23 nucleotides in length, have a duplex of about 19 base pairs and two single-stranded overhangs, and are generally RNA. These RNA fragments are then reported to direct the degradation of target nucleic acids such as mRNA or pre-mRNA. It has also been reported that Dicer is also involved in excising 21- and 22-nucleotide small temporal RNAs (stRNAs) involved in translational control from precursor RNAs having a conserved structure. Hutvagner et al 2001, Science, 293, 834. Those skilled in the art will recognize that RNAi can be mediated by single-stranded or double-stranded oligonucleotides that include a sequence that is complementary or substantially complementary to a target sequence (e.g., in a target mRNA). Thus, in some embodiments of the present disclosure, the single-stranded oligonucleotides described herein can act as RNAi agents; in some embodiments, the double-stranded oligonucleotides described herein can act as RNAi agents. In some embodiments, the RNAi reaction involves an endonuclease complex, commonly referred to as the RNA-induced silencing complex (RISC), which directs the cleavage of single-stranded mRNA complementary to the antisense strand of the siRNA. In some embodiments, RISC directs the cleavage of a target RNA complementary to the provided oligonucleotide, which can be used as a single-stranded RNAi agent. In some embodiments, the cleavage of the target RNA occurs in the middle of the region complementary to the siRNA duplex or the antisense strand of the single-stranded RNAi agent. In some embodiments, RNA interference is directed by a single-stranded oligonucleotide that acts as a single-stranded RNAi agent and can direct RNA interference in a mechanism involving the RISC pathway.

[0171] RNAi Agent: As used herein, the terms "RNAi agent", "iRNA agent", etc. refer to APOC3 oligonucleotides that reduce the level and / or activity (e.g., translation) of a target gene product (e.g., a transcript of a target gene, such as pre-mRNA or mRNA) when administered to a system in which the target gene product is being expressed or has been expressed. In some embodiments, the RNAi agent can be or comprise a single-stranded oligonucleotide or a double-stranded oligonucleotide. In some embodiments, the RNAi agent can have a structure recognized in the art, such as siRNA (short interfering RNA), shRNA (short or small hairpin RNA), dsRNA (double-stranded RNA), microRNA, etc. In some embodiments, the RNAi agent can specifically bind to an RNA target (e.g., a transcript of a target gene). In some embodiments, after binding to its target, the RNAi agent is loaded into RISC (RNA-induced silencing complex). In some embodiments, the RNAi agent directs the degradation of its target and / or inhibits the translation of its target through a mechanism involving the RISC (RNA-induced silencing complex) pathway in some embodiments. In some embodiments, the RNAi agent is an APOC3 oligonucleotide that activates the RISC complex / pathway. In some embodiments, the RNAi agent comprises an antisense strand sequence. In some embodiments, the RNAi agent comprises only one oligonucleotide strand (e.g., is a single-stranded oligonucleotide). In some embodiments, the single-stranded RNAi agent oligonucleotide can be or comprise a sense or antisense strand sequence, such as Sioud 2005 J. Mol. Biol. 348: 1079-1090. In some embodiments, the RNAi agent is a compound capable of directing RNA interference. In some embodiments, the RNAi agent can have a structure or format as present in a "canonical" siRNA structure. In some embodiments, the RNAi agent can have a structure different from the "canonical" siRNA structure. To name just a few examples, in some embodiments, the RNAi agent can be longer or shorter than canonical, can be blunt-ended, and / or can contain one or more modifications, mismatches, gaps, and / or nucleotide substitutions. In some embodiments, the RNAi agent contains a 3'-terminal cap as described in the present disclosure. Without wishing to be bound by any particular theory, the applicant proposes that in certain embodiments, the 3'-terminal cap can permit two functions: (1) permit RNA interference; and (2) increase the duration of activity and / or the biological half-life of the RNAi agent (which can be achieved, for example, by increasing binding to the PAZ domain of Dicer and / or one or more Ago proteins) and / or reduce or prevent degradation of the RNAi agent (e.g., by nucleases, such as those in serum or intestinal fluid). In some embodiments, the RNAi agent of the present disclosure targets (e.g., binds, anneals, etc.) the target mRNA.In some embodiments, exposure of an RNAi agent to its target results in a decrease in activity, level, and / or expression, such as “knockdown” or “knockout” of the target. In particular, in some embodiments, in the case of a disease, disorder, and / or condition characterized by overexpression and / or hyperactivity of a target gene, administration of an RNAi agent to a cell, tissue, or subject sufficiently knockdowns the target gene to restore normal activity levels or reduces the activity to a level at which the disease, disorder, and / or condition can be alleviated, ameliorated, mitigated, inhibited, prevented, the onset thereof delayed, the severity thereof reduced, and / or the occurrence of one or more of its symptoms or characteristics decreased. In some embodiments, the RNAi agent is double-stranded and comprises an antisense strand that is a single-stranded RNAi agent as described herein and that in combination with a sense strand can direct RNA interference.

[0172] Single-stranded RNA interference: As used herein, phrases such as “single-stranded RNAi” or “single-stranded RNA interference” refer to a process or method of gene silencing directed at least in part by administration of a single-stranded RNAi agent to a system (e.g., a cell, tissue, organ, subject, etc.), wherein the RNAi will be directed by the RNAi agent and requires the RISC pathway. The term may be used herein in some instances to distinguish from “double-stranded RNAi” or “double-stranded RNA interference,” wherein a double-stranded RNAi agent is administered to the system and may be further processed, e.g., such that one of its two strands is loaded into the RISC to, for example, inhibit translation, cleave target RNA, etc.

[0173] Single-stranded RNAi agent: As used herein, the phrase “single-stranded RNAi agent” refers to a single-stranded oligonucleotide that can direct single-stranded RNA interference (RNAi or iRNA) gene silencing via the RISC pathway. A single-stranded RNAi agent may comprise a polymer of one or more single-stranded nucleotides.

[0174] Subject: As used herein, the term “subject” or “test subject” refers to any organism to which a provided compound or composition is administered according to the present invention, e.g., for experimental, diagnostic, prophylactic, and / or therapeutic purposes. Typical subjects include animals (e.g., mammals such as mice, rats, rabbits, non-human primates, and humans; insects; worms; etc.) and plants. In some embodiments, the subject may be afflicted with and / or susceptible to a disease, disorder, and / or condition.

[0175] Basically: As used herein, the term "basically" refers to a qualitative situation that exhibits the target characteristic or property of an overall or near-overall range or degree. A base sequence that is basically complementary to a second sequence is not identical to the second sequence, but is mostly or nearly the same as the second sequence. Additionally, one of ordinary skill in the biological arts will understand that biological and chemical phenomena rarely (if ever) achieve complete and / or proceed to completion or achieve or avoid absolute results. Thus, the term "basically" is used herein to account for the lack of potential completeness inherent in many biological and / or chemical phenomena.

[0176] Affected: An individual who is "affected" with a disease, disorder, and / or condition has been diagnosed with and / or exhibits one or more symptoms of the disease, disorder, and / or condition.

[0177] Susceptible: An individual who is "susceptible" to a disease, disorder, and / or condition is an individual who has a higher risk of developing the disease, disorder, and / or condition than a member of the general public. In some embodiments, an individual who is susceptible to a disease, disorder, and / or condition may not have been diagnosed with the disease, disorder, and / or condition. In some embodiments, an individual who is susceptible to a disease, disorder, and / or condition may exhibit symptoms of the disease, disorder, and / or condition. In some embodiments, an individual who is susceptible to a disease, disorder, and / or condition may not exhibit symptoms of the disease, disorder, and / or condition. In some embodiments, an individual who is susceptible to a disease, disorder, and / or condition will develop the disease, disorder, and / or condition. In some embodiments, an individual who is susceptible to a disease, disorder, and / or condition will not develop the disease, disorder, and / or condition.

[0178] Systemic: As used herein, the phrases "systemic administration," "administered systemically," "peripheral administration," and "administered peripherally" have their understood meaning in the art and refer to the administration of a compound or composition such that it enters the system of a recipient.

[0179] Therapeutic agent: As used herein, the phrase "therapeutic agent" refers to any agent that has a therapeutic effect and / or elicits a desired biological and / or pharmacological effect when administered to a subject. In some embodiments, a therapeutic agent is any substance that can be used to alleviate, improve, mitigate, inhibit, prevent a disease, disorder, and / or condition, delay its onset, reduce its severity, and / or reduce the occurrence of one or more of its symptoms or characteristics.

[0180] Therapeutically effective amount: As used herein, the term "therapeutically effective amount" refers to the amount of a substance (e.g., a therapeutic agent, composition, and / or formulation) that elicits a desired biological response when administered as part of a treatment regimen. In some embodiments, a therapeutically effective amount of a substance is an amount sufficient to treat, diagnose, prevent, and / or delay the onset of a disease, disorder, and / or condition when administered to a subject afflicted with or susceptible to the disease, disorder, and / or condition. As will be appreciated by one of ordinary skill in the art, the effective amount of a substance can vary depending on factors such as the desired biological endpoint, the substance to be delivered, the target cell or tissue, etc. For example, an effective amount of a compound in a formulation for treating a disease, disorder, and / or condition is an amount that alleviates, improves, mitigates, inhibits, prevents the disease, disorder, and / or condition, delays its onset, reduces its severity, and / or reduces the occurrence of one or more of its symptoms or characteristics. In some embodiments, a therapeutically effective amount is administered as a single dose; in some embodiments, multiple unit doses are required to deliver a therapeutically effective amount.

[0181] Treat: As used herein, the term "treat", "treatment", or "treating" refers to any method used to partially or completely alleviate, improve, mitigate, inhibit, prevent a disease, disorder, and / or condition, delay its onset, reduce its severity, and / or reduce the occurrence of one or more of its symptoms or characteristics. Treatment can be administered to a subject who does not exhibit signs of a disease, disorder, and / or condition. In some embodiments, treatment can be administered to a subject who exhibits only early signs of a disease, disorder, and / or condition, for example, for the purpose of reducing the risk of developing lesions associated with the disease, disorder, and / or condition.

[0182] Unsaturated: As used herein, the term "unsaturated" means that a moiety has one or more unsaturated units.

[0183] Wild type: As used herein, the term "wild type" has its meaning as understood in the art and refers to an entity having a structure and / or activity as it exists in nature in a "normal" (as opposed to mutant, diseased, altered, etc.) state or background. One of ordinary skill in the art will appreciate that wild type genes and polypeptides often exist in multiple different forms (e.g., alleles).

[0184] Nucleic Acid: As used herein, the term "nucleic acid" includes any nucleotide and its polymers. As used herein, the term "polynucleotide" refers to polymeric forms of nucleotides of any length (ribonucleotides (RNA) or deoxyribonucleotides (DNA)). These terms refer to the primary structure of the molecule and thus include double-stranded and single-stranded DNA as well as double-stranded and single-stranded RNA. These terms include analogs of RNA or DNA made from modified nucleotides and / or modified polynucleotides (such as, but not limited to, methylated, protected, and / or capped nucleotides or polynucleotides) as equivalents. The terms cover polyribonucleotides or oligoribonucleotides (RNA) and polydeoxyribonucleotides or oligodeoxyribonucleotides (DNA); RNA or DNA derived from N-glycosides or C-glycosides of nucleobases and / or modified nucleobases; nucleic acids derived from sugars and / or modified sugars; and nucleic acids derived from phosphate bridges and / or modified internucleotide linkages. The terms cover nucleic acids containing any combination of nucleobases, modified nucleobases, sugars, modified sugars, phosphate bridges, or modified internucleotide linkages. Examples include, and are not limited to, nucleic acids containing a ribose moiety, nucleic acids containing a deoxyribose moiety, nucleic acids containing both ribose and deoxyribose moieties, and nucleic acids containing ribose and modified ribose moieties. Unless otherwise specified, the prefix poly generally refers to a nucleic acid containing 2 to about 10,000 nucleotide monomer units, and the prefix oligo refers to a nucleic acid containing 2 to about 200 nucleotide monomer units.

[0185] Nucleotide: As used herein, the term "nucleotide" refers to the monomer unit of a polynucleotide composed of a heterocyclic base, a sugar, and one or more internucleotide linkages. Naturally occurring bases (guanine (G), adenine (A), cytosine (C), thymine (T), and uracil (U)) are derivatives of purines or pyrimidines, but it is understood that naturally and non-naturally occurring base analogs are also included. Naturally occurring sugars are pentoses (five-carbon sugars) deoxyribose (which forms DNA) or ribose (which forms RNA), but it is understood that naturally and non-naturally occurring sugar analogs are also included. Nucleotides are linked by internucleotide linkages to form nucleic acids or polynucleotides. Many internucleotide linkages are known in the art (such as, but not limited to, phosphate esters, phosphorothioates, boranophosphates, etc.). Artificial nucleic acids include PNA (peptide nucleic acid), phosphotriesters, phosphorothioates, H-phosphonates, aminophosphates, boranophosphates, methylphosphonates, phosphonylacetates, thiophosphonylacetates, and other variants of the phosphate backbone of natural nucleic acids, such as those described herein. In some embodiments, natural nucleotides contain naturally occurring bases, sugars, and internucleotide linkages. As used herein, the term "nucleotide" also covers structural analogs used in place of natural or naturally occurring nucleotides, such as modified nucleotides and nucleotide analogs.

[0186] Modified nucleotide: The term "modified nucleotide" includes any chemical moiety that is structurally different from a natural nucleotide but capable of performing at least one function of a natural nucleotide. In some embodiments, the modified nucleotide comprises a modification at the sugar, base, and / or internucleoside linkage. In some embodiments, the modified nucleotide comprises a modified sugar, a modified nucleobase, and / or a modified internucleoside linkage. In some embodiments, the modified nucleotide is capable of having at least one function of a nucleotide, such as forming a subunit in a polymer that can base pair with a nucleic acid comprising at least a complementary base sequence.

[0187] Analogue: The term "analogue" means any functional analogue wherein the chemical moiety is structurally different from a reference chemical moiety or class of moieties but capable of performing at least one function of such reference chemical moiety or class of moieties. As a non-limiting example, a nucleotide analogue is structurally different from a nucleotide but performs at least one function of a nucleotide; a nucleobase analogue is structurally different from a nucleobase but performs at least one function of a nucleobase; and so on.

[0188] Nucleoside: The term "nucleoside" refers to a moiety wherein a nucleobase or modified nucleobase is covalently attached to a sugar or modified sugar.

[0189] Modified nucleoside: The term "modified nucleoside" refers to a moiety derived from or chemically similar to a natural nucleoside, but which contains a chemical modification that distinguishes it from the natural nucleoside. Non-limiting examples of modified nucleosides include those that contain a modification at the base and / or sugar. Non-limiting examples of modified nucleosides include those that have a 2'-modification at the sugar. Non-limiting examples of modified nucleosides also include abasic nucleosides (which lack a nucleobase). In some embodiments, the modified nucleoside is capable of having at least one function of a nucleoside, such as forming a moiety in a polymer that can base pair with a nucleic acid comprising at least a complementary base sequence.

[0190] Nucleoside analogue: The term "nucleoside analogue" refers to a chemical moiety that is chemically different from a natural nucleoside but capable of performing at least one function of a nucleoside. In some embodiments, the nucleoside analogue comprises an analogue of the sugar and / or an analogue of the nucleobase. In some embodiments, the modified nucleoside is capable of having at least one function of a nucleoside, such as forming a moiety in a polymer that can base pair with a nucleic acid comprising a complementary base sequence.

[0191] Sugar: The term "sugar" refers to monosaccharides or polysaccharides in closed and / or open forms. In some embodiments, the sugar is a monosaccharide. In some embodiments, the sugar is a polysaccharide. Sugars include, but are not limited to, ribose, deoxyribose, furanopentose, pyranopentose, and pyranose hexose moieties. As used herein, the term "sugar" also encompasses structural analogs used in place of conventional sugar molecules, such as diols, polymers forming the backbone of nucleic acid analogs, glycol nucleic acid ("GNA"), etc. As used herein, the term "sugar" also encompasses structural analogs used in place of natural or naturally occurring nucleotides, such as modified sugars and nucleotide sugars.

[0192] Modified sugar: The term "modified sugar" refers to a moiety that can replace a sugar. Modified sugars mimic the spatial arrangement, electronic properties, or some other physicochemical property of sugars.

[0193] Nucleobase: The term "nucleobase" refers to the part of a nucleic acid that is involved in the hydrogen bonding that causes one nucleic acid strand to bind to another complementary strand in a sequence-specific manner. The most common naturally occurring nucleobases are adenine (A), guanine (G), uracil (U), cytosine (C), and thymine (T). In some embodiments, the naturally occurring nucleobases are modified adenine, guanine, uracil, cytosine, or thymine. In some embodiments, the naturally occurring nucleobases are methylated adenine, guanine, uracil, cytosine, or thymine. In some embodiments, the nucleobase is a "modified nucleobase", e.g., a nucleobase other than adenine (A), guanine (G), uracil (U), cytosine (C), and thymine (T). In some embodiments, the modified nucleobase is methylated adenine, guanine, uracil, cytosine, or thymine. In some embodiments, the modified nucleobase mimics the spatial arrangement, electronic properties, or some other physicochemical property of a nucleobase and retains the property of forming a hydrogen bond that causes one nucleic acid strand to bind to another nucleic acid strand in a sequence-specific manner. In some embodiments, the modified nucleobase can pair with all five naturally occurring bases (uracil, thymine, adenine, cytosine, or guanine) with substantially no effect on the melting behavior, recognition by intracellular enzymes, or activity of the oligonucleotide duplex. As used herein, the term "nucleobase" also encompasses structural analogs used in place of natural or naturally occurring nucleotides, such as modified nucleobases and nucleobase analogs.

[0194] Modified nucleobase: The terms "modified nucleobase", "modified base", etc. refer to chemical moieties that are chemically different from a nucleobase but capable of performing at least one function of a nucleobase. In some embodiments, the modified nucleobase is a modified nucleobase-containing moiety. In some embodiments, the modified nucleobase is capable of having at least one function of a nucleobase, e.g., forming a moiety in a polymer that can base pair with a nucleic acid containing at least a complementary base sequence.

[0195] 3′-end cap: The term “3′-end cap” refers to a non-nucleotide chemical moiety that binds to the 3′-end of an APOC3 oligonucleotide (e.g., an RNAi agent). In some embodiments, the 3′-end cap replaces the 3′-terminal dinucleotide. In some embodiments, the 3′-end cap of an APOC3 oligonucleotide performs at least one of the following functions: allowing oligonucleotide-directed RNA interference, protecting the oligonucleotide from degradation or reducing the amount or rate of oligonucleotide degradation (e.g., by nucleases), reducing off-target effects of the sense strand, or increasing the activity, duration, or efficacy of oligonucleotide-directed RNA interference. By describing the 3′-end cap as “non-nucleotide,” it is meant that the 3′-end cap is not a nucleotide moiety or oligonucleotide moiety that is linked to the sugar moiety of the remainder of the APOC3 oligonucleotide as if it were part of the APOC3 oligonucleotide chain. Certain exemplary 3′-end caps are described herein. Those of ordinary skill in the art will understand that other 3′-end caps known in the art can be used in accordance with the present disclosure.

[0196] Blocking group: The term “blocking group” refers to a group that masks the reactivity of a functional group. The functional group can subsequently be de-masked by removing the blocking group. In some embodiments, the blocking group is a protecting group.

[0197] Moiety: The term “moiety” refers to a particular segment or functional group of a molecule. A chemical moiety is often recognized as a chemical entity that is incorporated into or attached to a molecule.

[0198] Solid support: The term “solid support” refers to any support that enables the synthesis of nucleic acids. In some embodiments, the term refers to a glass or polymer that is insoluble in the medium used to perform the reaction steps for synthesizing nucleic acids and is derivatized to contain reactive groups. In some embodiments, the solid support is highly cross-linked polystyrene (HCP) or controlled pore glass (CPG). In some embodiments, the solid support is controlled pore glass (CPG). In some embodiments, the solid support is a hybrid support of controlled pore glass (CPG) and highly cross-linked polystyrene (HCP).

[0199] Linker or linking moiety: The terms “linker,” “linking moiety,” etc. refer to any chemical moiety that links one chemical moiety to another. In some embodiments, the linker is a moiety that links one oligonucleotide to another oligonucleotide in a polymer. In some embodiments, the linker is a moiety that is optionally located between the terminal nucleoside and the solid support or between the terminal nucleoside and another nucleoside, nucleotide, or nucleic acid.

[0200] Gene: As used herein, the terms "gene", "recombinant gene", and "gene construct" refer to a DNA molecule or a portion of a DNA molecule that encodes a protein or a portion thereof. The DNA molecule may contain an open reading frame encoding a protein (such as an exon sequence), and may also include intron sequences. As used herein, the term "intron" refers to a DNA sequence that is present in a given gene, does not translate into a protein, and is present between exons in some but not all cases. It may be desirable for the gene to be operably linked to (or it may contain) one or more promoters, enhancers, repressors, and / or other regulatory sequences to regulate the activity or expression of the gene, as is well known in the art.

[0201] Complementary DNA: As used herein, "complementary DNA" or "cDNA" includes recombinant polynucleotides synthesized by reverse transcribing mRNA and from which intervening sequences (introns) have been removed.

[0202] Oligonucleotide: The term "oligonucleotide" refers to a polymer or oligomer of nucleotides and may contain any combination of natural and unnatural nucleobases, sugars, and internucleotide linkages.

[0203] Oligonucleotides can be single-stranded or double-stranded. As used herein, the term "oligonucleotide strand" encompasses single-stranded oligonucleotides. A single-stranded oligonucleotide may have double-stranded regions (formed by two portions of the single-stranded oligonucleotide), and a double-stranded oligonucleotide containing two oligonucleotide strands may have single-stranded regions, for example, at regions where the two oligonucleotide strands are not complementary to each other. In some embodiments, the oligonucleotide is capable of directing the reduction of the expression and / or level of a target gene or its gene product. In some embodiments, the oligonucleotide is capable of directing the reduction of the expression and / or level of a target gene or its gene product by RNA interference. In some embodiments, the oligonucleotide is capable of directing the reduction of the expression and / or level of a target gene or its gene product by a biochemical mechanism that does not involve RNA interference or RISC (including but not limited to RNase H-mediated knockdown or steric hindrance of gene expression). In some embodiments, the oligonucleotide is capable of directing the reduction of the expression and / or level of a target gene or its gene product by RNA interference and / or RNase H-mediated knockdown. Exemplary oligonucleotides include but are not limited to structural genes, genes including control and termination regions, self-replicating systems (such as viral or plasmid DNA), single-stranded and double-stranded RNAi agents and other RNA interference reagents (RNAi agents or iRNA agents), shRNA, antisense oligonucleotides, ribozymes, microRNAs, microRNA mimics, super microRNAs, aptamers, antisense microRNAs, microRNA antagonists, Ul linkers, triplex-forming oligonucleotides, G-quadruplex oligonucleotides, RNA activators, immunostimulatory oligonucleotides, and decoy oligonucleotides.

[0204] Double-stranded and single-stranded oligonucleotides that effectively induce RNA interference are also referred to herein as RNAi agents or iRNA agents. In some embodiments, these oligonucleotides that induce RNA interference associate with a cytoplasmic multi-protein complex called the RNAi-induced silencing complex (RISC). In many embodiments, double-stranded RNAi agents are long enough such that they can be cleaved by endogenous molecules (e.g., by Dicer), generating smaller oligonucleotides that can enter the RISC machinery and participate in the cleavage and / or translational inhibition of a target sequence (e.g., a target mRNA sequence) mediated by RISC.

[0205] The oligonucleotides of the present disclosure can have various lengths. In certain embodiments, the length of the oligonucleotide can range from about 2 to about 200 nucleotides. In various related embodiments, the lengths of single-stranded, double-stranded, and triple-stranded oligonucleotides can range from about 4 to about 10 nucleotides, about 10 to about 50 nucleotides, about 20 to about 50 nucleotides, about 15 to about 30 nucleotides, or about 20 to about 30 nucleotides. In some embodiments, the length of the APOC3 oligonucleotide is from about 10 to about 40 nucleotides. In some embodiments, the length of the APOC3 oligonucleotide is from about 9 to about 39 nucleotides. In some embodiments, the length of the oligonucleotide is at least 4 nucleotides. In some embodiments, the length of the oligonucleotide is at least 5 nucleotides. In some embodiments, the length of the oligonucleotide is at least 6 nucleotides. In some embodiments, the length of the oligonucleotide is at least 7 nucleotides. In some embodiments, the length of the oligonucleotide is at least 8 nucleotides. In some embodiments, the length of the oligonucleotide is at least 9 nucleotides. In some embodiments, the length of the oligonucleotide is at least 10 nucleotides. In some embodiments, the length of the oligonucleotide is at least 11 nucleotides. In some embodiments, the length of the oligonucleotide is at least 12 nucleotides. In some embodiments, the length of the oligonucleotide is at least 15 nucleotides. In some embodiments, the length of the oligonucleotide is at least 20 nucleotides. In some embodiments, the length of the oligonucleotide is at least 25 nucleotides. In some embodiments, the length of the oligonucleotide is at least 30 nucleotides. In some embodiments, the oligonucleotide is a duplex of complementary strands that are at least 18 nucleotides in length. In some embodiments, the oligonucleotide is a duplex of complementary strands that are at least 21 nucleotides in length. In some embodiments, each nucleotide counted in terms of length independently comprises an optionally substituted nucleobase selected from adenine, cytosine, guanosine, thymine, and uracil.

[0206] Inter-nucleotide linkage: As used herein, the phrase "inter-nucleotide linkage" generally refers to the linkage that connects the nucleoside units of an APOC3 oligonucleotide or nucleic acid. In some embodiments, the inter-nucleotide linkage is a phosphodiester linkage (natural phosphate linkage) such as that present in naturally occurring DNA and RNA molecules. In some embodiments, the term "inter-nucleotide linkage" includes modified inter-nucleotide linkages. In some embodiments, the inter-nucleotide linkage is a "modified inter-nucleotide linkage" in which each oxygen atom of the phosphodiester linkage is optionally and independently replaced by an organic or inorganic moiety. In some embodiments, such organic or inorganic moieties are selected from, but not limited to, =S, =Se, =NR', -SR', -SeR', -N(R')2, B(R')3, -S-, -Se- and -N(R')-, where each R' is independently defined and described as in the present disclosure. In some embodiments, the inter-nucleotide linkage is a phosphotriester linkage, a phosphorothioate diester linkage or a modified phosphorothioate triester linkage.

[0207] One of ordinary skill in the art will appreciate that due to the presence of acidic or basic moieties in the linkage, at a given pH, the inter-nucleotide linkage can exist as an anion or a cation.

[0208] In some embodiments, "all (Rp)" or "all (Sp)" is used to indicate that all chiral linked phosphorus atoms in the oligonucleotide have the same Rp or Sp configuration, respectively.

[0209] Oligonucleotide type: As used herein, the phrase "oligonucleotide type" is used to define an APOC3 oligonucleotide having a specific base sequence, backbone linkage pattern (i.e., inter-nucleotide linkage type pattern, such as phosphate, phosphorothioate, etc.), backbone chiral center pattern (i.e., linked phosphorus stereochemistry pattern (Rp / Sp)), and backbone phosphorus modification pattern (e.g., the "-XLR 1 " group pattern in Formula I). In some embodiments, oligonucleotides with a common designation of "type" are identical in structure to each other.

[0210] Those skilled in the art will appreciate that the synthesis methods of the present disclosure provide a degree of control during the synthesis of APOC3 oligonucleotide chains such that the nucleotide units of the oligonucleotide chain can be designed and / or selected in advance to have a specific stereochemistry at the linking phosphorus, and / or a specific modification at the linking phosphorus, and / or a specific base, and / or a specific sugar. In some embodiments, the APOC3 oligonucleotide chain is designed and / or selected in advance to have a specific combination of stereocenters at the linking phosphorus. In some embodiments, the APOC3 oligonucleotide chain is designed and / or determined to have a specific combination of modifications at the linking phosphorus. In some embodiments, the APOC3 oligonucleotide chain is designed and / or selected to have a specific combination of bases. In some embodiments, the APOC3 oligonucleotide chain is designed and / or selected to have a specific combination of one or more of the above structural features. In some embodiments, the present disclosure provides a composition comprising or consisting of a plurality of oligonucleotide molecules (e.g., a chirally controlled oligonucleotide composition). In some embodiments, all such molecules are of the same type (i.e., are identical to each other in structure). However, in many embodiments, the provided composition typically comprises a plurality of different types of oligonucleotides in a predetermined relative amount.

[0211] Chiral control: As used herein, "chiral control" refers to the control of the stereochemical design of the chiral linking phosphorus in the chiral internucleotide linkages within an APOC3 oligonucleotide. In some embodiments, the control is achieved through chiral elements that are absent from the sugar and base moieties of the APOC3 oligonucleotide. For example, in some embodiments, the control is achieved by using one or more chiral auxiliaries during the preparation of the oligonucleotide as shown in the present disclosure, and the chiral auxiliaries are typically part of the chiral phosphoramidites used in the oligonucleotide preparation. In contrast to chiral control, those of ordinary skill in the art will appreciate that if conventional oligonucleotide synthesis without the use of chiral auxiliaries is used to form chiral internucleotide linkages, such conventional oligonucleotide synthesis cannot control the stereochemistry at the chiral internucleotide linkages. In some embodiments, the stereochemical design of each chiral linking phosphorus in the chiral internucleotide linkages within the APOC3 oligonucleotide is controlled.

[0212] Chirality-Controlled Oligonucleotide Compositions: As used herein, the terms "chirality-controlled oligonucleotide composition", "chirality-controlled nucleic acid composition", etc. refer to a composition comprising a plurality of oligonucleotides (or nucleic acids) that share 1) a common base sequence, 2) a common backbone linkage pattern, and 3) a common backbone phosphorus modification pattern, wherein the plurality of oligonucleotides (or nucleic acids) have the same stereochemistry at one or more chiral internucleotide linkages (chirality-controlled internucleotide linkages), and the levels of the various oligonucleotides (or nucleic acids) in the composition are predetermined (e.g., by a chirality-controlled oligonucleotide article to form one or more chiral internucleotide linkages). In some embodiments, about 1% - 100% (e.g., about 5% - 100%, 10% - 100%, 20% - 100%, 30% - 100%, 40% - 100%, 50% - 100%, 60% - 100%, 70% - 100%, 80 - 100%, 90 - 100%, 95 - 100%, 50% - 90%, or about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%, or at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) of all the oligonucleotides in the chirality-controlled oligonucleotide composition are the plurality of oligonucleotides. In some embodiments, about 1% - 100% (e.g., about 5% - 100%, 10% - 100%, 20% - 100%, 30% - 100%, 40% - 100%, 50% - 100%, 60% - 100%, 70% - 100%, 80 - 100%, 90 - 100%, 95 - 100%, 50% - 90%, or about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%, or at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) of all the oligonucleotides in the chirality-controlled oligonucleotide composition that share a common base sequence are the plurality of oligonucleotides.In some embodiments, about 1%-100% (e.g., about 5%-100%, 10%-100%, 20%-100%, 30%-100%, 40%-100%, 50%-100%, 60%-100%, 70%-100%, 80-100%, 90-100%, 95-100%, 50%-90%, or about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) of all the oligonucleotides sharing a common base sequence, a common backbone linkage pattern, and a common backbone phosphorus modification pattern in a chiroselective oligonucleotide composition are the plurality of oligonucleotides. In some embodiments, the predetermined level is all the oligonucleotides in the composition, or all the oligonucleotides sharing a common base sequence in the composition (e.g., the plurality of oligonucleotides or APOC3 oligonucleotide type), or all the oligonucleotides sharing a common base sequence, a common backbone linkage pattern, and a common backbone phosphorus modification pattern as the plurality of oligonucleotides, or about 1%-100% (e.g., about 5%-100%, 10%-100%, 20%-100%, 30%-100%, 40%-100%, 50%-100%, 60%-100%, 70%-100%, 80-100%, 90-100%, 95-100%, 50%-90%, or about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) of all the oligonucleotides sharing a common base sequence, a common base modification pattern, a common sugar modification pattern, a common internucleotide linkage type pattern, and / or a common internucleotide linkage modification pattern in the composition.In some embodiments, multiple oligonucleotides share the same stereochemistry at about 1 - 50 (e.g., about 1 - 10, 1 - 20, 5 - 10, 5 - 20, 10 - 15, 10 - 20, 10 - 25, 10 - 30, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20, or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) chiral internucleotide linkages. In some embodiments, multiple oligonucleotides share the same stereochemistry at about 1% - 100% (e.g., about 5% - 100%, 10% - 100%, 20% - 100%, 30% - 100%, 40% - 100%, 50% - 100%, 60% - 100%, 70% - 100%, 80 - 100%, 90 - 100%, 95 - 100%, 50% - 90%, about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%, or up to 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99%) of chiral internucleotide linkages. In some embodiments, each chiral internucleotide linkage is a chirally controlled internucleotide linkage, and the composition is a fully chirally controlled oligonucleotide composition. In some embodiments, not all chiral internucleotide linkages are chirally controlled internucleotide linkages, and the composition is a partially chirally controlled oligonucleotide composition. In some embodiments, a chirally controlled oligonucleotide composition comprises a predetermined level of a single oligonucleotide or nucleic acid type. For example, in some embodiments, a chirally controlled oligonucleotide composition comprises one oligonucleotide type. In some embodiments, a chirally controlled oligonucleotide composition comprises more than one oligonucleotide type. In some embodiments, a chirally controlled oligonucleotide composition comprises multiple oligonucleotide types. In some embodiments, a chirally controlled oligonucleotide composition is a composition of oligonucleotides of oligonucleotide types, the composition comprising a predetermined level of oligonucleotides of multiple oligonucleotide types.

[0213] Chirally pure: As used herein, the phrase "chirally pure" is used to describe the relative amount of APOC3 oligonucleotides (such as single-stranded RNAi agents) in which all oligonucleotides are present in a single diastereomeric form with respect to the linked phosphorus.

[0214] Chirally homogeneous: As used herein, the phrase "chirally homogeneous" is used to describe an APOC3 oligonucleotide molecule or type in which all nucleotide units have the same stereochemistry at the linking phosphorus. For example, an APOC3 oligonucleotide in which all nucleotide units have the Rp stereochemistry at the linking phosphorus is chirally homogeneous. Similarly, an APOC3 oligonucleotide in which all nucleotide units have the Sp stereochemistry at the linking phosphorus is chirally homogeneous.

[0215] Predetermined: Predetermined means deliberately selected, e.g., as opposed to occurring randomly or being achieved without control. One of ordinary skill in the art reading this specification will appreciate that the present disclosure provides techniques that allow for the selection of specific chemical and / or stereochemical features to be incorporated into an oligonucleotide composition and also allows for the controlled preparation of oligonucleotide compositions having such chemical and / or stereochemical features. Such provided compositions are "predetermined" as described herein. Compositions that happen to contain certain oligonucleotides that have been produced by a process that cannot be controlled to deliberately produce specific chemical and / or stereochemical features are not "predetermined" compositions. In some embodiments, a predetermined composition is a composition that can be deliberately reproduced (e.g., by repeating a controlled process). In some embodiments, a predetermined level of multiple oligonucleotides in a composition means controlling the absolute amount and / or relative amount (ratio, percentage, etc.) of the multiple oligonucleotides in the composition. In some embodiments, a predetermined level of multiple oligonucleotides in a composition is achieved by chiral control of oligonucleotide preparation.

[0216] Linking phosphorus: As defined herein, the phrase "linking phosphorus" is used to indicate that the particular phosphorus atom being referred to is the phosphorus atom present in the internucleotide linkage, which corresponds to the phosphorus atom of the phosphodiester of the internucleotide linkage as present in naturally occurring DNA and RNA. In some embodiments, the linking phosphorus atom is in a modified internucleotide linkage, wherein each oxygen atom of the phosphodiester linkage is optionally and independently replaced by an organic or inorganic moiety. In some embodiments, the linking phosphorus atom is a P of formula I L . In some embodiments, the linking phosphorus atom is chiral.

[0217] P modification: As used herein, the term "P modification" refers to any modification at the linking phosphorus other than a stereochemical modification. In some embodiments, P modifications include addition, substitution, or removal of pendant moieties covalently attached to the linking phosphorus. In some embodiments, "P modification" is -X-L-R 1 , wherein each of X, L, and R 1 is independently as defined and described in the present disclosure.

[0218] For the purposes of the present disclosure, chemical elements are identified according to the CAS version of the Periodic Table of the Elements on the inside front cover of the Handbook of Chemistry and Physics, 67th Edition, 1986 - 87.

[0219] The methods and structures described herein with respect to the compounds and compositions of the present disclosure are also applicable to pharmaceutically acceptable acid or base addition salts and all stereoisomeric forms of these compounds and compositions. BRIEF DESCRIPTION OF THE DRAWINGS

[0220] FIG. 1. FIG. 1, including Figures 1A to 1L , shows a cartoon of various ssRNAi formats and hybridization formats.

[0221] 2. Figure 2 shows a cartoon of various antisense oligonucleotide formats.

[0222] FIG. 3. Figure 3A shows an exemplary polymer format. Oligonucleotides can be joined directly and / or via a linker. As shown, the polymer can comprise oligonucleotide monomers of the same or different structures / types. In some embodiments, the monomers of the polymer are ssRNAi agents. In some embodiments, the monomers of the polymer are ribonuclease H - dependent antisense oligonucleotides (ASOs). The monomers can be joined at various positions, such as the 5′ - terminus, 3′ - terminus, or an intermediate position. Figure 3B shows exemplary chemical methods for joining monomers, which can perform their functions via various pathways to form a polymer. DETAILED DESCRIPTION

[0223] Synthetic oligonucleotides provide molecular tools that are useful in a wide variety of applications. For example, oligonucleotides can be used in therapeutics, diagnostics, research, and novel nanomaterial applications. The use of naturally occurring nucleic acids (e.g., unmodified DNA or RNA) is limited, for example, by their susceptibility to endonucleases and exonucleases. Accordingly, various synthetic counterparts have been developed to circumvent these drawbacks. These include, in particular, synthetic oligonucleotides containing chemical modifications (e.g., base modifications, sugar modifications, backbone modifications, etc.), which render these molecules less susceptible to degradation and improve other properties of the oligonucleotides. From a structural perspective, modifications to the internucleoside phosphodiester linkages can introduce chirality, and certain properties of the oligonucleotides can be affected by the configuration of the phosphorus atoms that form the oligonucleotide backbone. For example, in vitro studies have shown that the properties of antisense oligonucleotides, such as binding affinity, sequence specificity for binding to complementary RNA, and stability to nucleases, are particularly affected by the chirality of the backbone phosphorus atoms.

[0224] The present disclosure particularly encompasses the recognition that structural elements of oligonucleotides, such as chemical modifications (e.g., modifications of sugars, bases, and / or internucleotide linkages) or patterns thereof, conjugation with lipids or other moieties, and / or stereochemistry [e.g., stereochemistry of backbone chiral centers (chiral internucleotide linkages) and / or patterns thereof], can have a significant impact on properties and activities (e.g., stability, specificity, selectivity, activity of reducing the level of target gene products (transcripts and / or proteins), etc.). In some embodiments, oligonucleotide properties can be modulated by optimizing chemical modifications (modifications of bases, sugars, and / or internucleotide linkage moieties), chemical modification patterns, stereochemistry, and / or stereochemistry patterns.

[0225] In some embodiments, the present disclosure demonstrates that oligonucleotide compositions comprising oligonucleotides having controlled structural elements (e.g., controlled chemical modifications and / or controlled backbone stereochemistry patterns) provide unexpected properties and activities, including but not limited to those described herein. In some embodiments, the provided compositions comprising oligonucleotides having chemical modifications (e.g., base modifications, sugar modifications, internucleotide linkage modifications, etc.) or patterns thereof have improved properties and activities. Non-limiting examples of such improved properties include: directing the expression and / or reduction of the level of a target gene or its gene product; and / or directing RNA interference; and / or directing RNase H-mediated knockdown. In some embodiments, the present disclosure provides techniques (e.g., oligonucleotides, compositions, methods, etc.) for single-stranded RNAi. In some embodiments, the provided oligonucleotides are ssRNAi agents.

[0226] In some embodiments, RNA interference is reported to be a post-transcriptional gene silencing technique that uses RNAi agents to target RNAs (such as gene transcripts like messenger RNA (mRNA)) containing sequences complementary to the RNAi agent for cleavage mediated by the RISC (RNA-induced silencing complex) pathway. Substantially, it is reported that one type of RNAi occurs when ribonuclease III (Dicer) cleaves long dsRNA (double-stranded RNA) (e.g., exogenous dsRNA introduced into mammalian cells) into shorter fragments called siRNA. siRNA (small interfering RNA or short inhibitory RNA) is typically about 21 to 23 nucleotides in length and contains an approximately 19-base pair duplex. It is reported that the smaller RNA fragments then mediate the degradation of the target mRNA. The RNAi response is also reported to be characterized by an endonuclease complex, commonly called the RNA-induced silencing complex (RISC), which directs the cleavage of single-stranded mRNA complementary to the antisense strand of the siRNA. It is reported that the cleavage of the target RNA occurs in the middle of the region complementary to the antisense strand of the siRNA duplex. Using RNAi agents for target transcripts is reported to result in a reduction in gene activity, level, and / or expression, such as "knockdown" or "knockout" of the target gene or target sequence. Artificial siRNA can be used both as a therapeutic agent and for experimental purposes.

[0227] On the one hand, RNA interference agents include single-stranded RNAs that interact with target RNA sequences to direct the cleavage of the target RNA. Without being bound by theory, it is reported that type III endonuclease called Dicer degrades long double-stranded RNAs introduced into plant and invertebrate cells into siRNA (Sharp et al., Genes Dev. 2001, 15:485). It is reported that a ribonuclease III-like enzyme, Dicer, processes dsRNA into short interfering RNAs of 19 - 23 base pairs, which are characterized by two-base 3′ overhangs (Bernstein et al., (2001) Nature 409:363). It is reported that the siRNA is then incorporated into the RNA-induced silencing complex (RISC), where one or more helicases unwind the siRNA duplex, enabling the complementary antisense strand to guide target recognition (Nykanen et al., (2001) Cell 107:309). After binding to the appropriate target mRNA, one or more endonucleases within the RISC cleave the target to induce silencing (Elbashir et al., (2001) Genes Dev. 15:188). Thus, on the one hand, the present disclosure relates to single-stranded RNAs that promote the formation of the RISC complex to achieve target gene silencing.

[0228] In some embodiments, according to the present disclosure, suitable RNAi agents can be selected by any method known in the art or any method that can be conceived by a person of ordinary skill in the art. For example, the selection criteria can include one or more of the following steps: preliminary analysis of the target gene sequence and design of the RNAi agent; such design can consider sequence similarity among species (human, cynomolgus monkey, mouse, etc.) and differences from other (non-target) genes; in vitro screening of the RNAi agent (e.g., at 10 nM in cells expressing the target transcript); determination of EC50 or IC50 in cells; determination of the viability of cells treated with the RNAi agent, wherein in some embodiments, the RNAi agent targeting the target does not inhibit the viability of these cells; testing with human PBMC (peripheral blood mononuclear cells), e.g., testing the level of TNF-α to estimate immunogenicity, wherein immunostimulatory sequences are generally less required; testing in a human whole blood assay, wherein fresh human blood is treated with the RNAi agent and cytokine / chemokine levels [e.g., TNF-α (tumor necrosis factor-α) and / or MCP1 (monocyte chemoattractant protein 1)] are determined, wherein immunostimulatory sequences are generally less required; determination of in vivo gene knockdown using cells or tumors in test animals; and optimization of specific modifications of the RNAi agent.

[0229] It has been reported that the so-called canonical siRNA structure is a double-stranded RNA molecule, wherein each strand is about 21 nucleotides in length. It has been reported that these two strands are the antisense (or "guide") strand, which recognizes and binds to the complementary sequence in the target transcript, and the sense (or "passenger") strand, which is complementary to the antisense strand. It has been reported that the sense strand and the antisense strand are largely complementary and generally form two 3′ overhangs of 2 nucleotides at both ends.

[0230] Although the canonical siRNA structure has been reported to be double-stranded, the RNAi agent can also be single-stranded. In some embodiments, the single-stranded RNAi agent corresponds to the antisense strand of the double-stranded siRNA, and the single-stranded RNAi agent lacks the corresponding passenger strand.

[0231] However, it has been reported that not all structural elements of the tested single-stranded RNAi agents are effective; it has been reported that introducing some structural elements into the APOC3 oligonucleotide can interfere with single-stranded RNA interference activity.

[0232] In some embodiments, the present disclosure provides oligonucleotides and compositions useful as RNAi agents. In some embodiments, the present disclosure provides oligonucleotides and compositions useful as single-stranded RNAi agents. The present disclosure particularly provides novel structures of single-stranded oligonucleotides capable of directing RNA interference. Without wishing to be bound by any particular theory, the present disclosure states that single-stranded RNAi agents have advantages over double-stranded RNAi agents. For example, the commercial cost of single-stranded RNAi agents is lower because only one strand needs to be constructed. Additionally or alternatively, only one strand (the antisense strand) is administered to target the target transcript. The source of dsRNA-directed off-target effects is the loading of the sense strand into RISC and the binding and knockdown of unwanted targets (Jackson et al. 2003 Nat. Biotech. 21: 635-637), and single-stranded RNAi agents can cause fewer off-target effects compared to the corresponding double-stranded RNAi agents. Additionally, some single-stranded RNAi agents, including some disclosed herein, can target specific sequences that have not been successfully targeted by double-stranded RNAi agents previously (e.g., they can significantly reduce the levels of the sequence and / or sequence products (transcript and / or protein) compared to double-stranded RNAi agents). The present disclosure particularly provides novel formats (modifications, stereochemistry, combinations thereof, etc.) of oligonucleotides capable of directing single-stranded RNA interference.

[0233] Oligonucleotide

[0234] In some embodiments, the provided oligonucleotides can direct a decrease in the expression and / or level of a target gene or its gene product. In some embodiments, the provided oligonucleotides can direct a decrease in the level of a target product. In some embodiments, the provided oligonucleotides can decrease the transcript level of a target gene. In some embodiments, the provided oligonucleotides can decrease the mRNA level of a target gene. In some embodiments, the provided oligonucleotides can decrease the protein level encoded by a target gene. In some embodiments, the provided oligonucleotides can direct a decrease in the expression and / or level of a target gene or its gene product via RNA interference. In some embodiments, the provided oligonucleotides can direct a decrease in the expression and / or level of a target gene or its gene product via biochemical mechanisms that do not involve RNA interference or RISC (including but not limited to RNase H-mediated knockdown or steric hindrance of gene expression). In some embodiments, the provided oligonucleotides can direct a decrease in the expression and / or level of a target gene or its gene product via RNA interference and / or RNase H-mediated knockdown. In some embodiments, the provided oligonucleotides can direct a decrease in the expression and / or level of a target gene or its gene product by sterically blocking translation upon binding to the target gene mRNA, and / or by altering or interfering with mRNA splicing and / or exon inclusion or exclusion. In some embodiments, the provided oligonucleotides comprise one or more structural elements described herein or known in the art according to the present disclosure, such as base sequence; modification; stereochemistry; internucleotide linkage pattern; backbone linkage pattern; backbone chiral center pattern; backbone phosphorus modification pattern; additional chemical moieties, including but not limited to one or more targeting moieties, lipid moieties, and / or carbohydrate moieties, etc.; seed region; post-seed region; 5′-end structure; 5′-end region; 5′ nucleotide moiety; 3′-end region; 3′-end dinucleotide; 3′-end cap; etc. In some embodiments, the seed region of the APOC3 oligonucleotide is or comprises the second to eighth, second to seventh, second to sixth, third to eighth, third to seventh, third to seventh, or fourth to eighth, or fourth to seventh nucleotides counted from the 5′ end; and the post-seed region of the oligonucleotide is the region immediately 3′ to the seed region and between the seed region and the 3′-end region.

[0235] In some embodiments, the provided composition comprises an APOC3 oligonucleotide. In some embodiments, the provided composition comprises one or more lipid moieties, one or more carbohydrate moieties (except as otherwise specified, other than the sugar moieties of the nucleoside units that form the oligonucleotide chain with internucleotide linkages), and / or one or more targeting components.

[0236] In some embodiments, the target sequence is a sequence that binds to an APOC3 oligonucleotide as described herein. In many embodiments, the target sequence is identical or exactly complementary to the provided oligonucleotide or the sequence of consecutive residues therein (e.g., the provided oligonucleotide includes a targeting binding sequence that is identical or exactly complementary to the target sequence). In some embodiments, a small number of differences / mismatches are tolerated between the APOC3 oligonucleotide (the relevant portion thereof) and its target sequence. In many embodiments, the target sequence is present within a target gene. In many embodiments, the target sequence is present in a transcript (e.g., mRNA and / or pre-mRNA) produced by the target gene.

[0237] According to the present disclosure, various linkers, lipid moieties, carbohydrate moieties, and targeting moieties can be used, including many known in the art. In some embodiments, the lipid moiety is a targeting moiety. In some embodiments, the carbohydrate moiety is a targeting moiety. In some embodiments, the targeting moiety is a lipid moiety. In some embodiments, the targeting moiety is a carbohydrate moiety. As will be readily appreciated by those skilled in the art, according to the present disclosure, various linkers, including those described herein, can be used to link two moieties, such as a lipid / carbohydrate / targeting component and an APOC3 oligonucleotide moiety. As will be readily appreciated by those skilled in the art, the linkers described for linking two moieties can also be used to link other moieties, e.g., a linker used to link a lipid and an APOC3 oligonucleotide moiety can also be used to link a carbohydrate or targeting moiety to the APOC3 oligonucleotide moiety, and vice versa.

[0238] In some embodiments, the present disclosure provides chiral-controlled oligonucleotides and oligonucleotide compositions. For example, in some embodiments, the provided composition contains a predetermined level of one or more individual oligonucleotide types, where the APOC3 oligonucleotide type is defined by: 1) base sequence; 2) backbone linkage pattern; 3) backbone chiral center pattern; and 4) backbone P modification pattern. In some embodiments, a particular oligonucleotide type can be defined by: 1A) base identity; 1B) base modification pattern; 1C) sugar modification pattern; 2) backbone linkage pattern; 3) backbone chiral center pattern; and 4) backbone P modification pattern. In some embodiments, oligonucleotides of the same oligonucleotide type are identical. In some embodiments, the present disclosure provides a chiral-controlled oligonucleotide composition of oligonucleotides, where the composition contains a predetermined level of a plurality of oligonucleotides, where the plurality of oligonucleotides share a common base sequence and contain the same linked phosphorus configuration at at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 chiral internucleotide linkages (chiral-controlled internucleotide linkages).

[0239] In some embodiments, the provided oligonucleotides comprise from 2 to 30 chirality-controlled internucleotide linkages. In some embodiments, the provided oligonucleotides comprise from 5 to 30 chirality-controlled internucleotide linkages. In some embodiments, the provided oligonucleotides comprise from 10 to 30 chirality-controlled internucleotide linkages. In some embodiments, the provided oligonucleotides comprise 1 chirality-controlled internucleotide linkage. In some embodiments, the provided oligonucleotides comprise 2 chirality-controlled internucleotide linkages. In some embodiments, the provided oligonucleotides comprise 3 chirality-controlled internucleotide linkages. In some embodiments, the provided oligonucleotides comprise 4 chirality-controlled internucleotide linkages. In some embodiments, the provided oligonucleotides comprise 5 chirality-controlled internucleotide linkages. In some embodiments, the provided oligonucleotides comprise 6 chirality-controlled internucleotide linkages. In some embodiments, the provided oligonucleotides comprise 7 chirality-controlled internucleotide linkages. In some embodiments, the provided oligonucleotides comprise 8 chirality-controlled internucleotide linkages. In some embodiments, the provided oligonucleotides comprise 9 chirality-controlled internucleotide linkages. In some embodiments, the provided oligonucleotides comprise 10 chirality-controlled internucleotide linkages. In some embodiments, the provided oligonucleotides comprise 11 chirality-controlled internucleotide linkages. In some embodiments, the provided oligonucleotides comprise 12 chirality-controlled internucleotide linkages. In some embodiments, the provided oligonucleotides comprise 13 chirality-controlled internucleotide linkages. In some embodiments, the provided oligonucleotides comprise 14 chirality-controlled internucleotide linkages. In some embodiments, the provided oligonucleotides have 15 chirality-controlled internucleotide linkages. In some embodiments, the provided oligonucleotides have 16 chirality-controlled internucleotide linkages. In some embodiments, the provided oligonucleotides have 17 chirality-controlled internucleotide linkages. In some embodiments, the provided oligonucleotides have 18 chirality-controlled internucleotide linkages. In some embodiments, the provided oligonucleotides have 19 chirality-controlled internucleotide linkages. In some embodiments, the provided oligonucleotides have 20 chirality-controlled internucleotide linkages.

[0240] In some embodiments, the provided oligonucleotides are monomers. In some embodiments, the provided oligonucleotides are P-modified monomers. In some embodiments, the provided oligonucleotides are stereoisomeric monomers. In some embodiments, the provided oligonucleotides are stereoisomeric monomers having the Rp configuration. In some embodiments, the provided oligonucleotides are stereoisomeric monomers having the Sp configuration.

[0241] In some embodiments, the provided oligonucleotides are alternating polymers. In some embodiments, the provided oligonucleotides are P-modified alternating polymers. In some embodiments, the provided oligonucleotides are stereoregular alternating polymers.

[0242] In some embodiments, the provided oligonucleotides are block polymers. In some embodiments, the provided oligonucleotides are P-modified block polymers. In some embodiments, the provided oligonucleotides are stereoregular block polymers.

[0243] In some embodiments, the provided oligonucleotides are spacer polymers.

[0244] In some embodiments, the provided oligonucleotides are skip polymers.

[0245] In some embodiments, the provided oligonucleotides are hemimers. In some embodiments, the hemimer is an APOC3 oligonucleotide in which the 5′-terminal or 3′-terminal region has a sequence with a structural feature not possessed by the remaining oligonucleotide. In some embodiments, the 5′-terminal or 3′-terminal region has or comprises 2 to 20 nucleotides. In some embodiments, the structural feature is a base modification. In some embodiments, the structural feature is a sugar modification. In some embodiments, the structural feature is a P-modification. In some embodiments, the structural feature is the stereochemistry of the chiral internucleotide linkage. In some embodiments, the structural feature is or comprises a base modification, a sugar modification, a P-modification, or the stereochemistry of the chiral internucleotide linkage or a combination thereof. In some embodiments, the hemimer is an APOC3 oligonucleotide in which each sugar moiety of the 5′-terminal region shares a common modification. In some embodiments, the hemimer is an APOC3 oligonucleotide in which each sugar moiety of the 3′-terminal region shares a common modification. In some embodiments, any other sugar moieties in the oligonucleotide do not share the common sugar modification of the 5′ or 3′-terminal region. In some embodiments, an exemplary hemimer is an APOC3 oligonucleotide that contains, in one terminal region, a nucleoside with a substituted or unsubstituted 2′-O-alkyl sugar modification, a bicyclic sugar modification, a β-D-ribonucleoside, or a β-D-deoxyribonucleoside (e.g., a 2′-MOE-modified nucleoside, and LNA TM or ENA TMa sequence of nucleosides having a different sugar moiety (such as a substituted or unsubstituted 2′-O-alkyl sugar-modified nucleoside, a bicyclic sugar-modified nucleoside, or a natural nucleoside) in another terminal region. In some embodiments, the provided oligonucleotides are a combination of one or more of a monomer, an alternating polymer, a block polymer, a spacer polymer, a semi-polymer, and a skip polymer. In some embodiments, the provided oligonucleotides are a combination of one or more of a monomer, an alternating polymer, a block polymer, a spacer polymer, and a skip polymer. For example, in some embodiments, the provided oligonucleotides are both an alternating polymer and a spacer polymer. In some embodiments, the provided nucleotides are both a spacer polymer and a skip polymer. Those skilled in the art of chemistry and synthesis will recognize that numerous other combinations of styles are available and are limited only by the commercial availability and / or synthetic accessibility of the components required to synthesize the oligonucleotides according to the methods of the present disclosure. In some embodiments, the semi-polymer structure provides advantageous benefits. In some embodiments, the provided oligonucleotide is a 5′-semi-polymer that contains a modified sugar moiety in the 5′-terminal sequence. In some embodiments, the provided oligonucleotide is a 5′-semi-polymer that contains a modified 2′-sugar moiety in the 5′-terminal sequence.

[0246] In some embodiments, the provided oligonucleotides contain one or more optionally substituted nucleotides. In some embodiments, the provided oligonucleotides contain one or more modified nucleotides. In some embodiments, the provided oligonucleotides contain one or more optionally substituted nucleosides. In some embodiments, the provided oligonucleotides contain one or more modified nucleosides. In some embodiments, the provided oligonucleotides contain one or more optionally substituted LNAs.

[0247] In some embodiments, the provided oligonucleotides contain one or more optionally substituted nucleobases. In some embodiments, the provided oligonucleotides contain one or more optionally substituted natural nucleobases. In some embodiments, the provided oligonucleotides contain one or more optionally substituted modified nucleobases. In some embodiments, the provided oligonucleotides contain one or more of 5-methylcytidine; 5-hydroxymethylcytidine, 5-formylcytosine, or 5-carboxycytosine. In some embodiments, the provided oligonucleotides contain one or more of 5-methylcytidine.

[0248] In some embodiments, each base (BA) is independently a nucleobase of optionally substituted or protected adenine, cytosine, guanosine, thymine, or uracil. As will be appreciated by those skilled in the art, various protected nucleobases, including those well known in the art, such as those used in oligonucleotide preparation (e.g., the protected nucleobases of WO / 2010 / 064146, WO / 2011 / 005761, WO / 2013 / 012758, WO / 2014 / 010250, US2013 / 0178612, WO / 2014 / 012081, WO / 2015 / 107425, WO2017 / 015555, and WO2017 / 062862, the protected nucleobases of each of which are incorporated herein by reference), and can be used in accordance with the present disclosure.

[0249] In some embodiments, the provided oligonucleotides comprise one or more optionally substituted sugars. In some embodiments, the provided oligonucleotides comprise one or more optionally substituted sugars found in naturally occurring DNA and RNA. In some embodiments, the provided oligonucleotides comprise one or more optionally substituted riboses or deoxyriboses. In some embodiments, the provided oligonucleotides comprise one or more optionally substituted riboses or deoxyriboses, wherein one or more hydroxyl groups of the ribose or deoxyribose moiety are optionally and independently replaced by: a halogen, R′, -N(R′)2, -OR′, or -SR′, where each R′ is independently defined as above and as described herein. In some embodiments, the provided oligonucleotides comprise one or more optionally substituted deoxyriboses, wherein the 2′ position of the deoxyribose is optionally and independently substituted by: a halogen, R′, -N(R′)2, -OR′, or -SR′, where each R′ is independently defined as above and as described herein. In some embodiments, the provided oligonucleotides comprise one or more optionally substituted deoxyriboses, wherein the 2′ position of the deoxyribose is optionally and independently substituted by a halogen. In some embodiments, the provided oligonucleotides comprise one or more optionally substituted deoxyriboses, wherein the 2′ position of the deoxyribose is optionally and independently substituted by one or more -F halogens. In some embodiments, the provided oligonucleotides comprise one or more optionally substituted deoxyriboses, wherein the 2′ position of the deoxyribose is optionally and independently substituted by -OR′, where each R′ is independently defined as above and as described herein. In some embodiments, the provided oligonucleotides comprise one or more optionally substituted deoxyriboses, wherein the 2′ position of the deoxyribose is optionally and independently substituted by -OR′, where each R′ is independently an optionally substituted C1-C6 aliphatic. In some embodiments, the provided oligonucleotides comprise one or more optionally substituted deoxyriboses, wherein the 2′ position of the deoxyribose is optionally and independently substituted by -OR′, where each R′ is independently an optionally substituted C1-C6 alkyl. In some embodiments, the provided oligonucleotides comprise one or more optionally substituted deoxyriboses, wherein the 2′ position of the deoxyribose is optionally and independently substituted by -OMe. In some embodiments, the provided oligonucleotides comprise one or more optionally substituted deoxyriboses, wherein the 2′ position of the deoxyribose is optionally and independently substituted by -O-methoxyethyl.

[0250] In some embodiments, the provided oligonucleotides are hybridized oligonucleotide strands. In certain embodiments, the provided oligonucleotides are partially hybridized oligonucleotide strands. In certain embodiments, the provided oligonucleotides are fully hybridized oligonucleotide strands. In certain embodiments, the provided oligonucleotides are double-stranded oligonucleotides. In certain embodiments, the provided oligonucleotides are triple-stranded oligonucleotides (e.g., triplexes).

[0251] In some embodiments, any structure of the APOC3 oligonucleotide depicted in WO2012 / 030683 can be modified according to the methods of the present disclosure to provide compositions with chiral control thereof. For example, in some embodiments, the composition with chiral control includes stereochemical control at any one or more chiral-linked phosphorus atoms, optionally by incorporation of one or more P modifications described in WO2012 / 030683 or the present disclosure. For example, in some embodiments, specific nucleotide units of the APOC3 oligonucleotide of WO2012 / 030683 are preselected to set stereochemical modifications at the linked phosphorus of the nucleotide unit and / or to perform P modification with chiral control at the linked phosphorus of the nucleotide unit.

[0252] In some embodiments, the provided oligonucleotides include nucleic acid analogs, such as, for example, GNA, LNA, PNA, TNA, F-HNA (F-THP or 3'-fluorotetrahydropyran), MNA (mannitol nucleic acid, for example, Leumann 2002 Bioorg. Med. Chem. 10: 841-854), ANA (anitol nucleic acid), and morpholino.

[0253] In some embodiments, the provided oligonucleotides are characterized as being capable of indirectly or directly increasing or decreasing the activity of a protein or inhibiting or promoting the expression of a protein. In some embodiments, the provided oligonucleotides are characterized in that they can be used to control cell proliferation, viral replication, and / or any other cell signaling process.

[0254] In some embodiments, the 5'-end and / or 3'-end of the provided oligonucleotides are modified. In some embodiments, the 5'-end and / or 3'-end of the provided oligonucleotides are modified with terminal cap moieties. Examples of such modifications, including terminal cap moieties, are described extensively herein and in the art, such as, for example, those described in U.S. Patent Application Publication US2009 / 0023675A1.

[0255] In some embodiments, oligonucleotides of the APOC3 oligonucleotide type, which are characterized by 1) a common base sequence and length, 2) a common backbone linkage pattern, and 3) a common backbone chiral center pattern, have the same chemical structure. For example, they have the same base sequence, the same nucleoside modification pattern, the same backbone linkage pattern (i.e., the pattern of internucleotide linkage types, such as phosphodiester, phosphorothioate, etc.), the same backbone chiral center pattern (i.e., the stereochemical pattern of linked phosphorus (Rp / Sp)), and the same backbone phosphorus modification pattern (e.g., the pattern of the "-XLR 1 " group in Formula I).

[0256] Single-stranded RNAi agents and antisense oligonucleotides

[0257] In some embodiments, the present disclosure provides oligonucleotides. In some embodiments, the present disclosure provides oligonucleotides that reduce the expression and / or level of a target gene or its gene product. Those of ordinary skill in the art reading the present disclosure will understand that, in some embodiments, the provided oligonucleotides can act as RNAi agents. Optionally or additionally, in some embodiments, the provided oligonucleotides can act via an RNase H-dependent mechanism and / or another biochemical mechanism that does not involve RNA interference.

[0258] The present disclosure particularly defines certain structural properties that may be particularly desirable and / or effective in APOC3 oligonucleotides. The present disclosure particularly defines certain structural properties that may be particularly desirable and / or effective in APOC3 oligonucleotides that act as RNAi agents. In some embodiments, the present disclosure defines certain structural properties that may be particularly desirable and / or effective in APOC3 oligonucleotides that act via an RNase H-dependent mechanism and / or other biochemical mechanisms. In some embodiments, the present disclosure defines certain structural properties that may be particularly desirable and / or effective in single-stranded ssRNAi agents (ssRNAi or ssRNAi agents); in some such embodiments, as further described below, such structural properties may be different from those that are particularly desirable and / or effective in the corresponding strand of a double-stranded RNAi agent (dsRNAi or dsRNAi agent). In some embodiments, the provided oligonucleotides are single-stranded RNAi agents (e.g., which can be loaded into RISC and / or can direct or enhance RISC-mediated targeting). In some embodiments, the provided oligonucleotides are antisense oligonucleotides (e.g., which can be loaded into RNase H and / or direct or enhance the cleavage of RNase H-mediated targets and / or operate via different biochemical mechanisms).

[0259] In some embodiments (including some single-stranded oligonucleotide embodiments), the oligonucleotides that act as RNAi agents can have one or more structural properties and / or functional properties that are different from those that act via an RNase H-dependent mechanism. In some embodiments, APOC3 oligonucleotides can direct a reduction in the expression and / or level of a target gene or its gene product by sterically blocking translation after binding to the target gene mRNA, and / or by altering or interfering with mRNA splicing and / or exon inclusion or exclusion (e.g., skipping). In some embodiments, APOC3 oligonucleotides can perform a function or a significant percentage of the function (e.g., 10%-100%, not less than 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or greater) independent of RNA interference or RISC.

[0260] In some embodiments, the provided oligonucleotide is an antisense oligonucleotide (ASO) that directs cleavage of a target RNA mediated by RNase H rather than RISC (RNA interference silencing complex).

[0261] In some embodiments, the provided oligonucleotide is a single-stranded RNAi (ssRNAi) agent that directs cleavage of a target mRNA mediated by RISC (RNA interference silencing complex) rather than the enzyme RNase H. In some embodiments, the APOC3 oligonucleotide can perform a function or a significant percentage of the function (e.g., 10%-100%, not less than 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or greater) independent of RNase H.

[0262] Double-stranded RNAi agents can also direct cleavage of a target mRNA using RISC rather than the enzyme RNase H. In some embodiments, a single-stranded RNAi agent differs from a double-stranded RNAi agent in that the ssRNAi agent contains only one oligonucleotide strand and generally does not contain a duplex region of significant length, while the dsRNAi agent contains a duplex region of significant length (e.g., at least about 15 bp or about 19 bp in a "canonical" siRNA). In some embodiments, the dsRNAi contains two separate complementary strands (which are not covalently linked) that form a duplex region (e.g., in a "canonical" siRNA), or a long single strand that contains two complementary sequences that together form a duplex region (e.g., in an shRNA or short hairpin RNA). In some embodiments of the dsRNAi, the passenger strand has a single-stranded nick that forms two strands. In some embodiments, the sequences and / or structural elements (chemical modifications, stereochemistry, etc.) required for a single-stranded RNAi agent shown to be effective in the present disclosure can be different from those required for an effective double-stranded RNAi agent.

[0263] The present disclosure particularly encompasses the recognition that certain designs (e.g., sequences and / or structural elements) that may be applicable to double-stranded RNAi agents may not be applicable to single-stranded RNAi agents (including single-stranded RNAi agents in the provided formats described herein), and vice versa. In some embodiments, the present disclosure provides designs for effective ssRNAi. In some embodiments, the present invention demonstrates that certain base sequences, when combined with structural elements according to the present disclosure (modifications, stereochemistry, one or more additional chemical moieties, etc.), can provide oligonucleotides with unexpectedly high activity, for example, when administered as ssRNAi agents, particularly compared to oligonucleotides containing the same sequence but double-stranded and administered as dsRNAi agents. In some embodiments, the present invention demonstrates that certain base sequences, when combined with structural elements according to the present disclosure (modifications, stereochemistry, one or more additional chemical moieties, etc.), can provide oligonucleotides with unexpectedly high activity, such as the ability to reduce the expression and / or level of a target gene or its gene product.

[0264] Structural and functional differences between single-stranded RNAi (ssRNAi) agents, double-stranded RNAi (dsRNAi) agents, and ribonuclease H-dependent antisense oligonucleotides (ASOs)

[0265] In some embodiments, single-stranded RNAi (ssRNAi) agents, double-stranded RNAi (dsRNAi) agents, and RNase H-dependent antisense oligonucleotides (ASOs) all involve the binding of an agent or oligonucleotide (or a portion thereof) to a complementary (or substantially complementary) target RNA (e.g., mRNA or pre-mRNA), followed by cleavage of the target RNA and / or reduction in the expression and / or level of the target gene or its gene product. In some embodiments, RNAi agents, whether double-stranded or single-stranded, utilize the RISC or RNA interference silencing complex that includes the enzyme Ago-2 (Argonaute-2). In some embodiments, the RNase H-dependent antisense oligonucleotide is single-stranded and utilizes a different enzyme, RNase H. RNase H is reported to be a cellular endonuclease that cleaves the RNA strand of an RNA:DNA duplex; see U.S. Patent No. 7,919,472. See also Saetrom (2004 Bioinformatics 20:3055-3063); Kretschmer-Kazemi Far et al. (2003 Nucleic Acids 31:4417-4424); Bertrand et al. (2002) Biochem. Biophys. Res. Comm. 296:1000-1004); Vickers et al. (2003 J. Biol. Chem. 278:7108). In some embodiments, oligonucleotides that can direct RNase H-mediated knockdown include, but are not limited to, those consisting of or comprising a region of contiguous 2'-deoxynucleotide units that do not contain a 2'-modification. In some embodiments, oligonucleotides that can direct RNase H-mediated knockdown are gapmer oligonucleotides or gapmers. In some embodiments, a gapmer comprises an internal region that contains a plurality of nucleotides that support RNase H cleavage and is located between outer regions that have a plurality of nucleotides that are chemically different from the nucleotides of the internal region. In some embodiments, a gapmer comprises 2'-deoxynucleotides that flank or are adjacent to one or both sides and do not contain a defined range of 2'-modifications. In some embodiments, the gap directs RNase H cleavage of the corresponding RNA target. In some embodiments, the flanks do not direct RNase H cleavage or serve as a substrate for RNase H cleavage. The flanks can have different lengths (including, but not limited to, 1 to 8 nt) and can include various modifications or analogs (including, but not limited to, 2'-modifications, including, but not limited to, 2'-OMe and 2'-MOE).As a non-limiting example, see U.S. Patent Nos. 9,550,988; 7,919,472; 5,013,830; 5,149,797; 5,220,007; 5,256,775; 5,366,878; 5,403,711; 5,491,133; 5,565,350; 5,623,065; 5,652,355; 5,652,356 and 5,700,922. In some embodiments, the presence of one or more such modifications or analogs may be associated with modified (e.g., increased, decreased, or altered) RNase H cleavage of the target.

[0266] In some embodiments, double-stranded RNAi agents, even their antisense strands, are structurally different from RNase H-dependent antisense oligonucleotides. In some embodiments, RNase H-dependent antisense oligonucleotides and siRNA oligonucleotides appear to have exactly opposite characteristics with respect to both 5′-end structure and overall duplex stability.

[0267] It has been reported that double-stranded RNAi agents can be naturally produced in cells by the Dicer enzyme, which can cleave larger RNA molecules (such as double-stranded RNA from invading viruses) into dsRNA. The canonical structure of a dsRNA agent includes two RNA strands, each about 19 to 23 nt in length, annealing the two RNA strands to form a double-stranded region of about 19-21 bp and two 3′ dinucleotide overhangs. For double-stranded RNAi agents, it has been reported that the sense strand has been unwound from the duplex before the antisense strand is incorporated into RISC. Except for the natural separation of double-stranded RNAi agents into antisense and sense strands, single-stranded RNAi agents have not been reported to be naturally produced in human cells.

[0268] The present disclosure particularly provides the following teachings: In many cases, single-stranded RNAi agents are not just the separated antisense strands of double-stranded RNAi agents, because for example, the antisense strands of effective dsRNAi agents may be far less effective than the dsRNAi agents, and ssRNAi agents may be far less effective than ssRNAi agents when formulated as dsRNAi agents (e.g., by annealing with the sense strand). In some embodiments, double-stranded and single-stranded RNAi agents differ in many important respects. The structural parameters of double-stranded RNAi agents are not necessarily reflected in single-stranded RNAi agents.

[0269] In some embodiments, the target sequences to which the present disclosure teachings apply to double-stranded RNAi agents may not apply to single-stranded RNAi agents, and vice versa. For example, in at least some cases, the single-stranded forms of double-stranded RNAi agents may be ineffective. As a non-limiting example, Table 46A shows the construction of several ssRNAi agents with sequences derived from dsRNAi. These dsRNAi-based ssRNAi are generally less effective than the corresponding dsRNAi.

[0270] In some embodiments, double-stranded and single-stranded RNAi agents also differ in their sensitivity to the incorporation of chirality-controlled internucleotide linkages. For example, Matranga et al. (2005 Cell 123: 607-620) reported that the introduction of a single Sp internucleotide linkage (e.g., a single Sp PS) into the sense strand of a double-stranded RNAi agent significantly reduced RISC assembly and RNA interference activity. In contrast, in some embodiments, the data presented herein surprisingly demonstrate that the incorporation of Sp internucleotide linkages (e.g., Sp PS) can perform two functions on single-stranded RNAi agents: (a) it increases stability against nucleases; and (b) it does not interfere with RNA interference activity. Many exemplary oligonucleotides can be used as effective single-stranded RNAi agents that contain one or more chirality-controlled internucleotide linkages (e.g., shown herein as Sp internucleotide linkages or Sp PS (phosphorothioate)).

[0271] Optionally or additionally, double-stranded and single-stranded RNAi agents can differ in terms of immunogenicity. In some embodiments, it has been reported that some single-stranded RNAi agents are more immunogenic than double-stranded RNAi agents. Sioud J. Mol. Biol. (2005) 348, 1079-1090. In some embodiments, it has been reported that several double-stranded RNAi agents do not induce an immune response, while the corresponding single-stranded RNAi agents do. In some embodiments, the present disclosure provides oligonucleotides with low immunogenicity. In some embodiments, such oligonucleotides can be used as ssRNAi agents.

[0272] The present disclosure particularly encompasses the recognition that the conventional design of certain single-stranded RNAi agents derived from double-stranded RNAi agents (including the base sequence) often fails to provide effective single-stranded RNAi agents. In some embodiments, the present disclosure surprisingly demonstrates that ssRNAi agents derived from the base sequence of an effective RNase H-dependent ASO can produce effective ssRNAi agents (see Table 46A).

[0273] In some embodiments, the present disclosure provides oligonucleotides that can be used as effective RNase H-dependent ASOs, which contain a region of 2'-deoxynucleotides without 2'-modification and are complementary or substantially complementary to an RNA sequence or a portion thereof. In some embodiments, the region can be, for example, a core sequence of about 10 nt, flanked on one or both sides by flanks, where the indicated flanks are chemically different from the core and can include, as non-limiting examples, 2'-modifications or internucleotide linkage modifications.

[0274] Oligonucleotide

[0275] In some embodiments, the provided oligonucleotides can direct a reduction in the expression and / or level of a target gene or its gene product. In some embodiments, the provided oligonucleotides can direct a reduction in the expression and / or level of a target gene or its gene product through RNA interference. In some embodiments, the provided oligonucleotides can direct a reduction in the expression and / or level of a target gene or its gene product through biochemical mechanisms that do not involve RNA interference or RISC (including but not limited to RNase H-mediated knockdown or steric hindrance of gene expression). In some embodiments, the provided oligonucleotides can direct a reduction in the expression and / or level of a target gene or its gene product through RNA interference and / or RNase H-mediated knockdown. In some embodiments, the provided oligonucleotides can direct a reduction in the expression and / or level of a target gene or its gene product by spatially blocking translation after binding to the target gene mRNA, and / or by altering or interfering with mRNA splicing and / or exon inclusion or exclusion.

[0276] In some embodiments, the provided oligonucleotides have the structural elements, or a format or portion thereof, described herein.

[0277] In some embodiments, the provided oligonucleotides capable of directing a reduction in the expression and / or level of a target gene or its gene product have the structural elements, or a format or portion thereof, described herein.

[0278] In some embodiments, the provided oligonucleotides capable of directing a reduction in the expression and / or level of a target gene or its gene product have the format of any of the oligonucleotides disclosed herein, such as the oligonucleotide formats disclosed in Table 1A or in a figure or table or elsewhere disclosed herein.

[0279] In some embodiments, the provided oligonucleotides have any of the formats shown in FIG. 1.

[0280] The data provided in this disclosure indicate that various oligonucleotides in various formats are capable of directing a reduction in the expression and / or level of a target gene or its gene product targeting any one of a variety of different sequences in a variety of different genes in a variety of different species; additional data generated support the efficacy of the disclosed formats and ssRNAi agents not shown.

[0281] In some embodiments, the provided oligonucleotides capable of directing RNase H-mediated knockdown have the structural elements, or a format or portion thereof, described herein.

[0282] In some embodiments, the provided oligonucleotides capable of directing RNase H-mediated knockdown have the format of any of the oligonucleotides disclosed herein, such as the oligonucleotide formats disclosed in Table 1A or in a figure or table or elsewhere disclosed herein.

[0283] In some embodiments, the provided oligonucleotides have any of the formats shown in FIG. 1.

[0284] The data provided by this disclosure indicate that various oligonucleotides of various formats are capable of directing RNase H-mediated knockdown of any one of a variety of different sequences in a variety of different genes in a variety of different species; additional data generated support the efficacy of the disclosed formats and ssRNAi agents not shown.

[0285] In some embodiments, the provided oligonucleotides capable of directing single-stranded RNA interference have the structural elements described herein or a format or portion thereof.

[0286] In some embodiments, the provided oligonucleotides capable of directing single-stranded RNA interference have the format of any of the oligonucleotides disclosed herein, such as the oligonucleotide formats disclosed in Table 1A or in the figures or tables or elsewhere disclosed herein.

[0287] In some embodiments, the provided single-stranded RNAi agents have any of the formats shown in FIG. 1.

[0288] The data provided by this disclosure indicate that various RNAi agents of various formats are capable of directing RNA interference of any one of a variety of different sequences in any of a variety of different genes; additional data generated support the efficacy of the disclosed formats and ssRNAi agents not shown.

[0289] In some embodiments, the target of RNAi is a transcript. In some embodiments, the transcript is a pre-mRNA. In some embodiments, the transcript is a mature RNA. In some embodiments, the transcript is an mRNA. In some embodiments, the transcript contains a mutation. In some embodiments, the mutation is a frameshift. In some embodiments, the transcript contains a premature stop codon. In some embodiments, the target of RNAi is an RNA that is not an mRNA. In some embodiments, the target of RNAi is a non-coding RNA. In some embodiments, the target of RNAi is a long non-coding RNA. In some embodiments, the provided oligonucleotides in the provided compositions, such as the first plurality of oligonucleotides, comprise base modifications, sugar modifications, and / or internucleotide linkage modifications. In some embodiments, the provided oligonucleotides comprise base modifications and sugar modifications. In some embodiments, the provided oligonucleotides comprise base modifications and internucleotide linkage modifications. In some embodiments, the provided oligonucleotides comprise sugar modifications and internucleotide modifications. In some embodiments, the provided compositions comprise base modifications, sugar modifications, and internucleotide linkage modifications. Exemplary chemical modifications, such as base modifications, sugar modifications, internucleotide linkage modifications, etc., are well known in the art and include, but are not limited to, those described in the present disclosure. In some embodiments, the modified base is a substituted A, T, C, G, or U. In some embodiments, the sugar modification is a 2'-modification. In some embodiments, the 2'-modification is a 2'-F modification. In some embodiments, the 2'-modification is a 2'-OR1. In some embodiments, the 2'-modification is a 2'-OR1, where R1 is an optionally substituted alkyl. In some embodiments, the 2'-modification is a 2'-OMe. In some embodiments, the 2'-modification is a 2'-MOE. In some embodiments, the modified sugar moiety is a bridged bicyclic or polycyclic. In some embodiments, the modified sugar moiety is a bridged bicyclic or polycyclic having 5-20 ring atoms, where one or more of the ring atoms are optionally and independently heteroatoms. Exemplary ring structures are well known in the art, such as those present in BNA, LNA, etc. In some embodiments, the provided oligonucleotides comprise both one or more modified internucleotide linkages and one or more native phosphodiester linkages. In some embodiments, oligonucleotides and compositions thereof that simultaneously comprise modified internucleotide linkages and native phosphodiester linkages provide improved properties, such as activity, etc. In some embodiments, the modified internucleotide linkage is a chiral internucleotide linkage. In some embodiments, the modified internucleotide linkage is a phosphorothioate linkage. In some embodiments, the modified internucleotide linkage is a substituted phosphorothioate linkage.

[0290] The present disclosure particularly encompasses the recognition that stereorandom oligonucleotide products contain multiple different chemical entities that differ from each other in the stereochemical structure of various backbone chiral centers, for example, within the oligonucleotide chain. Without controlling the stereochemistry of the backbone chiral centers, stereorandom oligonucleotide products provide an uncontrolled composition that contains undefined levels of oligonucleotide stereoisomers. Even though these stereoisomers may have the same base sequence, they are different chemical entities at least due to their different backbone stereochemistry, and as demonstrated herein, they can have different properties, such as activity, etc. The present disclosure particularly provides new compositions that are or contain specific stereoisomers of a target oligonucleotide. In some embodiments, a specific stereoisomer can be defined, for example, by its base sequence, its length, its backbone linkage pattern, and its backbone chiral center pattern. As understood in the art, in some embodiments, the base sequence can refer to the identity and / or modification status of nucleoside residues in an APOC3 oligonucleotide (e.g., sugar and / or base moieties, relative to standard naturally occurring nucleotides such as adenine, cytosine, guanosine, thymine, and uracil) and / or the hybridization characteristics of such residues (i.e., the ability to hybridize to specific complementary residues). In some embodiments, the present disclosure provides an APOC3 oligonucleotide composition that contains a predetermined level of oligonucleotides of a single oligonucleotide type that are chemically identical, e.g., they have the same base sequence, the same nucleoside modification pattern (modifications of the sugar and base moieties, if any), the same backbone chiral center pattern, and the same backbone phosphorus modification pattern. The present invention particularly demonstrates that the individual stereoisomers of a specific oligonucleotide can exhibit different stabilities and / or activities from each other. In some embodiments, the property improvement achieved by including and / or positioning specific chiral structures within an APOC3 oligonucleotide can be comparable or even better than the property improvement achieved by using specific backbone linkages, residue modifications, etc. (e.g., by using certain types of modified phosphates [e.g., phosphorothioates, substituted phosphorothioates, etc.], sugar modifications [e.g., 2′-modifications, etc.], and / or base modifications [e.g., methylation, etc.]). The present disclosure particularly recognizes that in some embodiments, the properties (e.g., activity, etc.) of an APOC3 oligonucleotide can be regulated by optimizing its backbone chiral center pattern, optionally in combination with the modulation / optimization of one or more other features of the oligonucleotide (e.g., linkage pattern, nucleoside modification pattern, etc.). As illustrated by various examples in the present disclosure, the provided chiral-controlled oligonucleotide compositions can exhibit improved properties, such as improved single-stranded RNA interference activity, RNase H-mediated knockdown, improved delivery, etc.

[0291] In some embodiments, oligonucleotide properties can be regulated by optimizing stereochemistry (backbone chiral center pattern) and chemical modifications (modifications of bases, sugars, and / or internucleotide linkages) or their patterns.

[0292] In some embodiments, the common backbone chiral center patterns (e.g., the backbone chiral center patterns in single-stranded RNAi agents) include the following patterns: OSOSO, OSSSO, OSSSOS, SOSO, SOSO, SOSOS, SOSOSO, SOSOSOSO, SOSSSO, SSOSSSOSS, SSSOSOSSS, SSSSSOSOSSSS, SSSSS, SSSSSS, SSSSSSS, SSSSSSSS, SSSSSSSSS or RRR, where S represents a phosphorothioate in the Sp configuration, and O represents a phosphodiester, and where R represents a phosphorothioate in the Rp configuration.

[0293] In some embodiments, the achiral center is a phosphodiester linkage. In some embodiments, the chiral center in the Sp configuration is a phosphorothioate linkage. In some embodiments, the achiral center is a phosphodiester linkage. In some embodiments, the chiral center in the Sp configuration is a phosphorothioate linkage.

[0294] In some embodiments, the provided oligonucleotides comprise any of the stereochemical patterns described herein. In some embodiments, the provided oligonucleotides comprise any of the stereochemical patterns described herein and are capable of directing RNA interference. In some embodiments, the provided oligonucleotides comprise any of the stereochemical patterns described herein and are capable of directing RNase H-mediated knockdown. In some embodiments, the provided oligonucleotides comprise any of the stereochemical patterns described herein and are capable of directing both RNA interference and RNase H-mediated knockdown. In some embodiments, the provided oligonucleotides comprise any of the stereochemical patterns described herein and are capable of directing RNA interference, wherein the stereochemical pattern is in the seed and / or post-seed region. In some embodiments, the provided oligonucleotides comprise any of the stereochemical patterns described herein and are capable of directing both RNA interference and RNase H-mediated knockdown, wherein the stereochemical pattern is in the seed and / or post-seed region.

[0295] In some embodiments, the provided oligonucleotides comprise any of the modifications or patterns of modifications described herein. In some embodiments, the provided oligonucleotides comprise any of the modifications or patterns of modifications described herein and are capable of directing RNA interference. In some embodiments, the provided oligonucleotides comprise any of the patterns of modifications described herein and are capable of directing RNase H-mediated knockdown. In some embodiments, the provided oligonucleotides comprise any of the patterns of modifications described herein and are capable of directing both RNA interference and RNase H-mediated knockdown. In some embodiments, the provided oligonucleotides comprise any of the patterns of modifications described herein and are capable of directing RNA interference, wherein the pattern of modification is in the seed and / or post-seed region. In some embodiments, the provided oligonucleotides comprise any of the patterns of modifications described herein and are capable of directing both RNA interference and RNase H-mediated knockdown, wherein the pattern of modification is in the seed and / or post-seed region. In some embodiments, the modification or pattern of modification is a modification or pattern of modification at the 2′ position of the sugar. In some embodiments, the modification or pattern of modification is a modification of the sugar, such as at the 2′ position of the sugar, including but not limited to 2′-deoxy, 2′-F, 2′-OMe, 2′-MOE, and 2′-OR1, where R1 is an optionally substituted C1-6 alkyl.

[0296] In some embodiments, the present disclosure demonstrates, inter alia, that 2′-F modifications can improve single-stranded RNA interference. In some embodiments, the present disclosure demonstrates, inter alia, that Sp internucleotide linkages at the 5′- and 3′-termini can improve oligonucleotide stability. In some embodiments, the present disclosure demonstrates that, among other things, native phosphodiester linkages and / or Rp internucleotide linkages can improve the removal of oligonucleotides from the system. As will be appreciated by those of ordinary skill in the art, according to the present disclosure, various assays known in the art can be used to evaluate such properties.

[0297] In some embodiments, the provided oligonucleotides capable of directing single-stranded RNA interference comprise one or more modified sugar moieties. In some embodiments, 5% or more of the sugar moieties of the provided oligonucleotides are modified.

[0298] In some embodiments, the present disclosure provides an APOC3 oligonucleotide composition comprising a first plurality of oligonucleotides, wherein:

[0299] the first plurality of oligonucleotides have the same base sequence; and

[0300] the first plurality of oligonucleotides comprise one or more modified sugar moieties, or comprise one or more native phosphodiester linkages and one or more modified internucleotide linkages.

[0301] In some embodiments, the first plurality of oligonucleotides comprises one or more modified sugar moieties. In some embodiments, the provided oligonucleotides comprise one or more modified sugar moieties.

[0302] In some embodiments, the provided composition alters single-stranded RNA interference of a transcript such that an unwanted target and / or biological function is inhibited. In some embodiments, in such cases, the provided composition may also induce cleavage of the transcript after hybridization.

[0303] In some embodiments, each oligonucleotide of the first plurality of oligonucleotides comprises one or more modified sugar moieties and / or one or more modified internucleotide linkages. In some embodiments, each oligonucleotide of the first plurality of oligonucleotides comprises no more than about 95% unmodified sugar moieties. In some embodiments, each oligonucleotide of the first plurality of oligonucleotides comprises no more than about 50% unmodified sugar moieties. In some embodiments, each oligonucleotide of the first plurality of oligonucleotides comprises no more than about 5% unmodified sugar moieties. In some embodiments, each sugar moiety of the first plurality of oligonucleotides is independently modified.

[0304] In some embodiments, each oligonucleotide of the first plurality of oligonucleotides comprises two or more modified internucleotide linkages. In some embodiments, each oligonucleotide of the first plurality of oligonucleotides comprises three or more modified internucleotide linkages. In some embodiments, each oligonucleotide of the first plurality of oligonucleotides comprises four or more modified internucleotide linkages. In some embodiments, each oligonucleotide of the first plurality of oligonucleotides comprises five or more modified internucleotide linkages. In some embodiments, each oligonucleotide of the first plurality of oligonucleotides comprises ten or more modified internucleotide linkages.

[0305] In some embodiments, each oligonucleotide of the first plurality of oligonucleotides comprises no more than about 30% native phosphate linkages. In some embodiments, each oligonucleotide of the first plurality of oligonucleotides comprises no more than about 20% native phosphate linkages. In some embodiments, each oligonucleotide of the first plurality of oligonucleotides comprises no more than about 10% native phosphate linkages. In some embodiments, each oligonucleotide of the first plurality of oligonucleotides comprises no more than about 5% native phosphate linkages.

[0306] In some embodiments, the provided oligonucleotides comprise an increased level of one or more isotopes. In some embodiments, the provided oligonucleotides are labeled with one or more isotopes of one or more elements (e.g., hydrogen, carbon, nitrogen, etc.). In some embodiments, the provided oligonucleotides in the provided composition, e.g., the first plurality of oligonucleotides, comprise base modifications, sugar modifications, and / or internucleotide linkage modifications, wherein the oligonucleotides comprise an enriched level of deuterium. In some embodiments, the provided oligonucleotides are deuterium-labeled at one or more positions (substituting - 1 H for - 2 H). In some embodiments, one or more 1 H of the APOC3 oligonucleotide or any moiety conjugated to the oligonucleotide (e.g., targeting moiety, lipid moiety, etc.) is replaced by 2 H. Such oligonucleotides can be used in any of the compositions or methods described herein.

[0307] The present invention includes all pharmaceutically acceptable isotopically labeled compounds, wherein one or more atoms are replaced by atoms having the same atomic number but an atomic mass or mass number different from the atomic mass or mass number typically found in nature.

[0308] Examples of isotopes suitable for inclusion in the compounds of the present invention include isotopes of the following: hydrogen, such as 2 H and 3 H; carbon, such as 11 C, 13 C, and 14 C; chlorine, such as 36 Cl; fluorine, such as 18 F; iodine, such as 123 I, 124 I, and 125 I; nitrogen, such as 13 N and 15 N; oxygen, such as 15 O, 17 O, and 18 O; phosphorus, such as 32 p; and sulfur, such as 35 S.

[0309] Certain isotopically labeled compounds of formula (I), e.g., those incorporating a radioactive isotope, can be used in drug and / or substrate tissue distribution studies. The radioactive isotopes tritium (i.e., 3 H) and carbon-14 (i.e., 14 C) are particularly useful for this purpose in view of their ease of incorporation and ready means of detection.

[0310] Isotopically labeled with a heavier isotope such as deuterium (i.e., 2H) Substitution can provide certain therapeutic advantages arising from greater metabolic stability (e.g., increased in vivo half-life or reduced dosage requirements) and may thus be preferred in some cases.

[0311] Substitution with a positron-emitting isotope (such as 11 C, 18 F, 15 O and 13 N) can be used in positron emission tomography (PET) studies for examining substrate receptor occupancy.

[0312] Isotope-labeled compounds can generally be prepared by conventional techniques known to those skilled in the art or by processes similar to those described in the appended examples and preparations using appropriately isotope-labeled reagents in place of the previously used unlabeled reagents.

[0313] The compounds of the present invention may contain asymmetric or chiral centers and thus exist in different stereoisomeric forms. Unless otherwise specified, all stereoisomeric forms of the compounds of the present invention and their mixtures (including racemic mixtures) are intended to form part of the present invention. In addition, the present invention encompasses all geometric and positional isomers. For example, if the compounds of the present invention incorporate double bonds or fused rings, the cis and trans forms as well as mixtures are encompassed within the scope of the present invention.

[0314] The chiral compounds of the present invention (and their chiral precursors) can be obtained in enantiomerically enriched form using chromatography (usually high-pressure liquid chromatography (HPLC) or supercritical fluid chromatography (SFC)) on a resin having an asymmetric stationary phase and a mobile phase consisting of a hydrocarbon (usually heptane or hexane) containing from 0% to 50% isopropanol (usually 2% to 20%) and from 0% to 5% alkylamine (usually 0.1% diethylamine (DEA) or isopropylamine). The concentrated eluate gives an enriched mixture.

[0315] They can be separated into their individual diastereoisomers based on the physicochemical differences of the diastereoisomer mixture by methods well known to those skilled in the art, such as by chromatography and / or fractional crystallization. Enantiomers can be separated by reacting the enantiomer mixture with a suitable optically active compound (e.g., a chiral auxiliary such as a chiral alcohol or Mosher's acyl chloride) to convert the enantiomer mixture into a diastereoisomer mixture, separating the diastereoisomers and converting the individual diastereoisomers (e.g., by hydrolysis) into the corresponding pure enantiomers. Chiral HPLC columns can also be used to separate enantiomers. Optionally, the designated stereoisomers can be synthesized by using optically active starting materials, by asymmetric synthesis using optically active reagents, substrates, catalysts or solvents, or by asymmetric transformation of one stereoisomer into another.

[0316] In some embodiments, manipulating the structural elements of the control oligonucleotide, such as chemical modifications (e.g., modifications of the sugar, base, and / or internucleotide linkage), or patterns thereof, changes in stereochemistry (e.g., the stereochemistry of backbone chiral internucleotide linkages), or patterns thereof, substitution of atoms with isotopes of the same element, and / or conjugation with additional chemical moieties (e.g., lipid moieties, targeting moieties, etc.), can have a significant impact on the desired biological effect. In some embodiments, the desired biological effect is enhanced by more than 2-fold.

[0317] In some embodiments, the desired biological effect is to direct the reduction of the expression and / or level of a target gene or its gene product. In some embodiments, the desired biological effect is improved single-stranded RNA interference. In some embodiments, the desired biological effect is improved RNase H-mediated knockdown. In some embodiments, the desired biological effect is improved single-stranded RNA interference and / or RNase H-mediated knockdown.

[0318] In some embodiments, the present disclosure provides an APOC3 oligonucleotide composition comprising a first plurality of oligonucleotides, the first plurality of oligonucleotides:

[0319] 1) having a common base sequence complementary to a target sequence in a transcript; and

[0320] 2) comprising one or more modified sugar moieties and modified internucleotide linkages.

[0321] In some embodiments, the provided oligonucleotide composition is characterized in that when it contacts a transcript in a single-stranded RNA interference system, the RNAi-mediated knockdown of the transcript is improved compared to the knockdown observed under reference conditions, the reference conditions being selected from the group consisting of the absence of the composition, the presence of a reference composition, and combinations thereof.

[0322] In some embodiments, the present disclosure provides an APOC3 oligonucleotide composition comprising a first plurality of oligonucleotides capable of directing single-stranded RNA interference, wherein the APOC3 oligonucleotide type is defined by:

[0323] 1) the base sequence;

[0324] 2) the backbone linkage pattern;

[0325] 3) the backbone chiral center pattern; and

[0326] 4) the backbone phosphorus modification pattern,

[0327] The composition is chirally controlled because it is enriched in oligonucleotides of the particular oligonucleotide type relative to a substantially racemic product of oligonucleotides having the same base sequence.

[0328] The oligonucleotide composition is characterized in that when it contacts a transcript in a single-stranded RNA interference system, the RNAi-mediated knockdown of the transcript is improved compared to the knockdown observed under reference conditions selected from the group consisting of the absence of the composition, the presence of a reference composition, and combinations thereof.

[0329] In some embodiments, each of the consecutive nucleoside units is independently before and / or after a modified internucleoside linkage. In some embodiments, each of the consecutive nucleoside units is independently before and / or after a phosphorothioate linkage. In some embodiments, each of the consecutive nucleoside units is independently before and / or after a chirally controlled modified internucleoside linkage. In some embodiments, each of the consecutive nucleoside units is independently before and / or after a chirally controlled phosphorothioate bond linkage. In some embodiments, the modified internucleoside linkage has the structure of Formula I. In some embodiments, the modified internucleoside linkage has the structure of Formula I-a.

[0330] In some embodiments, the present disclosure provides a single-stranded RNAi agent comprising a predetermined level of a first plurality of oligonucleotides, wherein:

[0331] the first plurality of oligonucleotides have the same base sequence;

[0332] the first plurality of oligonucleotides comprise a seed region having 2, 3, 4, 5, 6, 7 or more consecutive Sp-modified internucleoside linkages, and a post-seed region having 2, 3, 4, 5, 6, 7, 8, 9, 10 or more consecutive Sp-modified internucleoside linkages.

[0333] In some embodiments, the seed region comprises 2 or more consecutive Sp-modified internucleoside linkages.

[0334] In some embodiments, the modified internucleoside linkage has the structure of Formula I. In some embodiments, the modified internucleoside linkage has the structure of Formula I-a.

[0335] As demonstrated by the present disclosure, in some embodiments, the provided oligonucleotide composition is characterized in that when it contacts a transcript in a single-stranded RNA interference system, the RNAi-mediated knockdown of the transcript is improved compared to the knockdown observed under reference conditions selected from the group consisting of the absence of the composition, the presence of a reference composition, and combinations thereof.

[0336] In some embodiments, the present disclosure provides an APOC3 oligonucleotide composition comprising a first plurality of oligonucleotides, the first plurality of oligonucleotides being defined by having:

[0337] 1) a common base sequence and length;

[0338] 2) a common backbone linkage pattern; and

[0339] 3) a common backbone chiral center pattern, the composition being a substantially pure preparation of a single oligonucleotide in that a predetermined level of the oligonucleotides in the composition have a common base sequence and length, a common backbone linkage pattern, and a common backbone chiral center pattern.

[0340] In some embodiments, the common base sequence and length may be referred to as a common base sequence. In some embodiments, oligonucleotides having a common base sequence may have the same nucleoside modification pattern, such as sugar modification, base modification, etc. In some embodiments, the nucleoside modification pattern may be represented by a combination of position and modification. In some embodiments, the backbone linkage pattern includes the position and type of each internucleotide linkage (e.g., phosphate, phosphorothioate, substituted phosphorothioate, etc.). The backbone chiral center pattern of the APOC3 oligonucleotide may be specified by the combination of the stereochemistry (Rp / Sp) of the linking phosphorus from 5′ to 3′. As exemplified above, the position of the achiral linkage may be obtained, for example, from the backbone linkage pattern.

[0341] As will be understood by those of ordinary skill in the art, a stereorandom or racemic preparation of an oligonucleotide is prepared by the diastereoselective and / or low stereoselective coupling of nucleotide monomers, typically without the use of any chiral auxiliaries, chiral modifying reagents, and / or chiral catalysts. In some embodiments, in a substantially racemic (or chirally uncontrolled) preparation of an oligonucleotide, all or most of the coupling steps are not chirally controlled because no coupling steps are specifically carried out to provide enhanced stereoselectivity. An exemplary substantially racemic preparation of an oligonucleotide is a preparation of a phosphorothioate oligonucleotide, which is prepared by sulfurizing the phosphite triester synthesized from common phosphoramidite oligonucleotides with diethyltetramine disulfide or (TETD) or 1,1-dioxo-3H-1,2-benzodithiol-3-one (BDTD) (a method well known in the art). In some embodiments, a substantially racemic preparation of an oligonucleotide provides a substantially racemic oligonucleotide composition (or chirally uncontrolled oligonucleotide composition).

[0342] As will be understood by those of ordinary skill in the art, in some embodiments, the diastereoselectivity of coupling or linkage can be evaluated by the diastereoselectivity of dimer formation under the same or comparable conditions, where the dimer has the same 5'- and 3'-nucleosides and internucleotide linkages.

[0343] In some embodiments, the present disclosure provides a chirally controlled oligonucleotide composition of a first plurality of oligonucleotides, because the composition is enriched in oligonucleotides of a single oligonucleotide type relative to a substantially racemic preparation of the same oligonucleotides. In some embodiments, the present disclosure provides a chirally controlled oligonucleotide composition of a first plurality of oligonucleotides, because the composition is enriched in oligonucleotides of a single oligonucleotide type relative to a substantially racemic preparation of the same oligonucleotides, which share:

[0344] 1) A common base sequence and length;

[0345] 2) A common backbone linkage pattern; and

[0346] 3) A common backbone chiral center pattern.

[0347] In some embodiments, the present disclosure provides an APOC3 oligonucleotide composition comprising a first plurality of oligonucleotides capable of directing single-stranded RNA interference, wherein the oligonucleotides belong to a specific oligonucleotide type, which is characterized by:

[0348] 1) A common base sequence and length;

[0349] 2) A common backbone linkage pattern; and

[0350] 3) A common backbone chiral center pattern;

[0351] The composition is chirally controlled because it is enriched in oligonucleotides of the specific oligonucleotide type relative to a substantially racemic preparation of oligonucleotides having the same base sequence and length.

[0352] In some embodiments, oligonucleotides having a common base sequence and length, a common backbone linkage pattern, and a common backbone chiral center pattern have a common backbone phosphorus modification pattern and a common base modification pattern. In some embodiments, oligonucleotides having a common base sequence and length, a common backbone linkage pattern, and a common backbone chiral center pattern have a common backbone phosphorus modification pattern and a common nucleoside modification pattern. In some embodiments, oligonucleotides having a common base sequence and length, a common backbone linkage pattern, and a common backbone chiral center pattern have the same structure.

[0353] In some embodiments, oligonucleotides of the APOC3 oligonucleotide type have a common backbone phosphorus modification pattern and a common sugar modification pattern. In some embodiments, oligonucleotides of the APOC3 oligonucleotide type have a common backbone phosphorus modification pattern and a common base modification pattern. In some embodiments, oligonucleotides of the APOC3 oligonucleotide type have a common backbone phosphorus modification pattern and a common nucleoside modification pattern. In some embodiments, oligonucleotides of the APOC3 oligonucleotide type are identical.

[0354] In some embodiments, oligonucleotides having a common base sequence and length, a common backbone linkage pattern, and a common backbone chiral center pattern have a common backbone phosphorus modification pattern. In some embodiments, oligonucleotides having a common base sequence and length, a common backbone linkage pattern, and a common backbone chiral center pattern have a common backbone phosphorus modification pattern and a common nucleoside modification pattern. In some embodiments, oligonucleotides having a common base sequence and length, a common backbone linkage pattern, and a common backbone chiral center pattern have a common backbone phosphorus modification pattern and a common sugar modification pattern. In some embodiments, oligonucleotides having a common base sequence and length, a common backbone linkage pattern, and a common backbone chiral center pattern have a common backbone phosphorus modification pattern and a common base modification pattern. In some embodiments, oligonucleotides having a common base sequence and length, a common backbone linkage pattern, and a common backbone chiral center pattern have a common backbone phosphorus modification pattern and a common nucleoside modification pattern. In some embodiments, oligonucleotides having a common base sequence and length, a common backbone linkage pattern, and a common backbone chiral center pattern are identical.

[0355] In some embodiments, the oligonucleotides in the provided composition have a common backbone phosphorus modification pattern. In some embodiments, the common base sequence is the base sequence of the APOC3 oligonucleotide type. In some embodiments, the provided composition is a chirally controlled APOC3 oligonucleotide composition because the composition contains a predetermined level of a first plurality of oligonucleotides of a single oligonucleotide type, wherein the APOC3 oligonucleotide type is defined by:

[0356] 1) base sequence;

[0357] 2) backbone linkage pattern;

[0358] 3) backbone chiral center pattern; and

[0359] 4) backbone phosphorus modification pattern.

[0360] As noted above and as understood in the art, in some embodiments, the base sequence of an APOC3 oligonucleotide can refer to the identity and / or modification status of nucleoside residues in the oligonucleotide (e.g., sugar and / or base moieties, relative to standard naturally occurring nucleotides such as adenine, cytosine, guanosine, thymine, and uracil) and / or the hybridization characteristics of such residues (i.e., the ability to hybridize to specific complementary residues).

[0361] In some embodiments, certain types of oligonucleotides are the same because they have the same base sequence (including length), the same chemical modification pattern of the sugar and base moieties, the same backbone linkage pattern (e.g., pattern of natural phosphate linkages, phosphorothioate linkages, phosphorothioate triesters, and combinations thereof), the same backbone chiral center pattern (e.g., stereochemical pattern of chiral internucleotide linkages (Rp / Sp)), and the same backbone phosphorus modification pattern (e.g., modification pattern of the phosphorus atom between nucleotides, such as -S- and -L-R of Formula I 1 ).

[0362] The present disclosure particularly recognizes that combinations of oligonucleotide structural elements (e.g., chemical modification patterns, backbone linkages, backbone chiral centers, and / or backbone phosphorus modifications) can provide surprisingly improved properties, such as biological activity.

[0363] In some embodiments, the provided chirally controlled (and / or stereochemically pure) article is an RNAi agent oligonucleotide.

[0364] In some embodiments, the provided chirally controlled (and / or stereochemically pure) article has an oligonucleotide comprising one or more modified backbone linkages, bases, and / or sugars.

[0365] In some embodiments, the provided compositions have oligonucleotides comprising one or more residues modified at the sugar moiety. In some embodiments, the provided compositions have oligonucleotides comprising one or more residues modified at the 2′ position of the sugar moiety (referred to herein as “2′-modifications”). Examples of such modifications are described above and herein and include, but are not limited to, 2′-OMe, 2′-MOE, 2′-LNA, 2′-F, FRNA, FANA, S-cEt, etc. In some embodiments, the provided compositions have oligonucleotides comprising one or more 2′-modified residues. For example, in some embodiments, the provided oligonucleotides comprise one or more residues that are 2′-O-methoxyethyl (2′-MOE)-modified residues. In some embodiments, the provided compositions have oligonucleotides that do not comprise any 2′-modifications. In some embodiments, the provided compositions are oligonucleotides that do not comprise any 2′-MOE residues. That is, in some embodiments, the provided oligonucleotides are not MOE-modified. The present disclosure describes additional exemplary sugar modifications.

[0366] In some embodiments, one or more is one. In some embodiments, one or more is two. In some embodiments, one or more is three. In some embodiments, one or more is four. In some embodiments, one or more is five. In some embodiments, one or more is six. In some embodiments, one or more is seven. In some embodiments, one or more is eight. In some embodiments, one or more is nine. In some embodiments, one or more is ten. In some embodiments, one or more is at least one. In some embodiments, one or more is at least two. In some embodiments, one or more is at least three. In some embodiments, one or more is at least four. In some embodiments, one or more is at least five. In some embodiments, one or more is at least six. In some embodiments, one or more is at least seven. In some embodiments, one or more is at least eight. In some embodiments, one or more is at least nine. In some embodiments, one or more is at least ten.

[0367] In some embodiments, the sugar moiety without 2′-modification is the sugar moiety present in natural DNA nucleosides.

[0368] One of ordinary skill in the art understands that various regions of the target transcript can be targeted by the provided compositions and methods. In some embodiments, the base sequence of the provided oligonucleotide contains an intron sequence. In some embodiments, the base sequence of the provided oligonucleotide contains an exon sequence. In some embodiments, the base sequence of the provided oligonucleotide contains both intron and exon sequences.

[0369] As one of ordinary skill in the art understands, the provided oligonucleotides and compositions can particularly target a large number of nucleic acid polymers. For example, in some embodiments, the provided oligonucleotides and compositions can target transcripts of nucleic acid sequences, where the common base sequence of the oligonucleotide (e.g., the base sequence of the APOC3 oligonucleotide type) includes or is a sequence complementary to the sequence of the transcript. In some embodiments, the common base sequence includes a sequence complementary to the sequence of the target. In some embodiments, the common base sequence is a sequence complementary to the sequence of the target. In some embodiments, the common base sequence includes or is a sequence 100% complementary to the sequence of the target. In some embodiments, the common base sequence includes a sequence 100% complementary to the sequence of the target. In some embodiments, the common base sequence is a sequence 100% complementary to the sequence of the target.

[0370] I

[0371] In some embodiments, the common base sequence includes or is a sequence complementary to a characteristic sequence element. In some embodiments, the common base sequence includes a sequence complementary to the characteristic sequence element. In some embodiments, the common base sequence is a sequence complementary to the characteristic sequence element. In some embodiments, the common base sequence includes or is a sequence 100% complementary to the characteristic sequence element. In some embodiments, the common base sequence includes a sequence 100% complementary to the characteristic sequence element. In some embodiments, the common base sequence is a sequence 100% complementary to the characteristic sequence element. In some embodiments herein, non-limiting examples of the characteristic sequence element are the seed region, the post-seed region, or a part of the seed region, or a part of the post-seed region, or the 3′-terminal dinucleotide.

[0372] In some embodiments, the characteristic sequence element includes or is a mutation. In some embodiments, the characteristic sequence element includes a mutation. In some embodiments, the characteristic sequence element is a mutation. In some embodiments, the characteristic sequence element includes or is a point mutation. In some embodiments, the characteristic sequence element includes a point mutation. In some embodiments, the characteristic sequence element is a point mutation. In some embodiments, the characteristic sequence element includes or is a SNP. In some embodiments, the characteristic sequence element includes a SNP. In some embodiments, the characteristic sequence element is a SNP.

[0373] In some embodiments, the common base sequence is 100% complementary to the target sequence and not 100% complementary to a sequence similar to the target sequence.

[0374] The present disclosure particularly recognizes that base sequences can affect oligonucleotide properties. In some embodiments, when an oligonucleotide having a base sequence is used to inhibit a target, for example, via a ribonuclease H-mediated pathway, the base sequence can affect the cleavage pattern of the target: for example, structurally similar (all phosphorothioate-linked, all stereorandom) oligonucleotides having different sequences can have different cleavage patterns.

[0375] In some embodiments, the common base sequence is a base sequence that includes an SNP.

[0376] As will be appreciated by those of ordinary skill in the art, the provided oligonucleotide compositions and methods have various uses known to those of ordinary skill in the art. Methods for evaluating the provided compositions and their properties and uses are also well known and practiced by those of ordinary skill in the art. Exemplary properties, uses, and / or methods include, but are not limited to, those described in WO / 2014 / 012081 and WO / 2015 / 107425.

[0377] In some embodiments, the chiral internucleotide linkage has the structure of Formula I. In some embodiments, the chiral internucleotide linkage is a phosphorothioate. In some embodiments, each chiral internucleotide linkage in a single oligonucleotide of the provided composition independently has the structure of Formula I. In some embodiments, each chiral internucleotide linkage in a single oligonucleotide of the provided composition independently is a phosphorothioate.

[0378] In some embodiments, the oligonucleotides of the present disclosure include one or more modified sugar moieties. In some embodiments, the oligonucleotides of the present disclosure include one or more modified base moieties. As known to those of ordinary skill in the art and as described in the present disclosure, various modifications can be introduced into the sugar and / or moieties. For example, in some embodiments, the modifications are those described in US9006198, WO2014 / 012081, and WO / 2015 / 107425, the sugar and base modifications of each of which are incorporated herein by reference.

[0379] In some embodiments, the sugar modification is a 2′-modification. Common 2′-modifications include, but are not limited to, 2′-OR 1 , where R 1Not hydrogen. In some embodiments, the modification is 2′-OR, where R is an optionally substituted aliphatic. In some embodiments, the modification is 2′-OMe. In some embodiments, the modification is 2′-O-MOE. In some embodiments, the present disclosure demonstrates that the inclusion and / or position of certain chiral-pure internucleotide linkages can provide comparable or better stability improvements as compared to those obtained by using modified backbone linkages, bases, and / or sugars. In some embodiments, the individual oligonucleotides provided in the compositions provided have no modification on the sugar. In some embodiments, the individual oligonucleotides provided in the compositions provided have no modification at the 2′-position of the sugar (i.e., the two groups at the 2′-position are -H / -H or -H / -OH). In some embodiments, the individual oligonucleotides provided in the compositions provided do not have any 2′-MOE modifications.

[0380] In some embodiments, the 2′-modification is -O-L- or -L-, which connects the 2′-carbon of the sugar moiety to another carbon of the sugar moiety. In some embodiments, the 2′-modification is -O-L- or -L-, which connects the 2′-carbon of the sugar moiety to the 4′-carbon of the sugar moiety. In some embodiments, the 2′-modification is S-cEt. In some embodiments, the modified sugar moiety is an LNA moiety.

[0381] In some embodiments, the 2′-modification is -F. In some embodiments, the 2′-modification is FANA. In some embodiments, the 2′-modification is FRNA.

[0382] In some embodiments, the sugar modification is a 5′-modification, e.g., R-5′-Me, S-5′-Me, etc.

[0383] In some embodiments, the sugar modification changes the size of the sugar ring. In some embodiments, the sugar modification is the sugar moiety in FHNA.

[0384] In some embodiments, the sugar modification replaces the sugar moiety with another cyclic or acyclic moiety. Examples of such moieties are well known in the art and include, but are not limited to, those used in morpholino (optionally having its phosphorodiamidate linkage), glycol nucleic acid, etc.

[0385] In some embodiments, the ssRNAi agent is or comprises an APOC3 oligonucleotide selected from the group consisting of any ssRNAi in any format as described in FIG. 1 or elsewhere herein. Those skilled in the art reading this specification will understand that the present disclosure expressly does not exclude the possibility that any oligonucleotide labeled herein as an ssRNAi agent may also or alternatively act by another mechanism (e.g., as an antisense oligonucleotide; mediated knockdown via the RNase H mechanism; steric hindrance of translation; or any other biochemical mechanism).

[0386] In some embodiments, the antisense oligonucleotide (ASO) is or comprises an APOC3 oligonucleotide selected from the group consisting of Figure 2 any oligonucleotide in any format as described. Those skilled in the art reading this specification will understand that the present disclosure expressly does not exclude the possibility that any oligonucleotide labeled herein as an antisense oligonucleotide (ASO) may also or alternatively act by another mechanism (e.g., using RISC as ssRNAi); the present disclosure also states that various ASOs can act by different mechanisms (using RNase H, sterically blocking translation or other post-transcriptional processes, altering the conformation of the target nucleic acid, etc.).

[0387] In some embodiments, the hybrid oligonucleotide is or comprises a group consisting of

[0388] APOC3 oligonucleotides: WV-2111, WV-2113, WV-2114, WV-2148, WV-2149, WV-2152, WV-2153, WV-2156, WV-2157, WV-2387, WV-3069, WV-7523, WV-7524, WV-7525, WV-7526, WV-7527, WV-7528, and Figure 1L formats S40 to S42; or formats 30-32, 66-69 of FIG. 1

[0389] any oligonucleotide of any one of 101-103. Those skilled in the art reading this specification will understand that the present disclosure expressly does not exclude the possibility that any oligonucleotide labeled herein as a hybrid oligonucleotide may also or alternatively act by another mechanism (e.g., as an antisense oligonucleotide; mediated knockdown via the RNase H mechanism; steric hindrance of translation; or any other biochemical mechanism).

[0390] Chirality-controlled oligonucleotides and chirality-controlled oligonucleotide compositions

[0391] In some embodiments, the provided oligonucleotides are capable of directing a reduction in the expression and / or level of a target gene or its gene product. In some embodiments, the provided oligonucleotides are capable of directing a reduction in the expression and / or level of a target gene or its gene product via RNA interference. In some embodiments, the provided oligonucleotides are capable of directing a reduction in the expression and / or level of a target gene or its gene product via a biochemical mechanism that does not involve RNA interference or RISC (including but not limited to RNase H-mediated knockdown or steric hindrance of gene expression). In some embodiments, the provided oligonucleotides are capable of directing a reduction in the expression and / or level of a target gene or its gene product via RNA interference and / or RNase H-mediated knockdown. In some embodiments, the provided oligonucleotides are capable of directing a reduction in the expression and / or level of a target gene or its gene product by sterically blocking translation upon annealing of the target gene mRNA, and / or by altering or interfering with mRNA splicing and / or exon inclusion or exclusion. In some embodiments, the provided oligonucleotides are chirally controlled.

[0392] The present disclosure provides chirally controlled oligonucleotides and chirally controlled oligonucleotide compositions having high crude purity and high diastereomeric purity. In some embodiments, the present disclosure provides chirally controlled oligonucleotides and chirally controlled oligonucleotide compositions having high crude purity. In some embodiments, the present disclosure provides chirally controlled oligonucleotides and chirally controlled oligonucleotide compositions having high diastereomeric purity.

[0393] In some embodiments, the single-stranded RNAi agent is a substantially pure article of the APOC3 oligonucleotide type, as oligonucleotides in the composition that are not of the oligonucleotide type are impurities formed during the preparation of the oligonucleotide type, in some cases, following certain purification procedures.

[0394] In some embodiments, the present disclosure provides oligonucleotides comprising one or more internucleotide linkages that are diastereomerically pure with respect to a chiral linked phosphorus. In some embodiments, the present disclosure provides oligonucleotides comprising one or more diastereomerically pure internucleotide linkages having the structure of Formula I. In some embodiments, the present disclosure provides oligonucleotides comprising one or more internucleotide linkages that are diastereomerically pure with respect to a chiral linked phosphorus and one or more phosphodiester linkages. In some embodiments, the present disclosure provides oligonucleotides comprising one or more diastereomerically pure internucleotide linkages having the structure of Formula I and one or more phosphodiester linkages. In some embodiments, the present disclosure provides oligonucleotides comprising one or more diastereomerically pure internucleotide linkages having the structure of Formula I-c and one or more phosphodiester linkages. In some embodiments, such oligonucleotides are prepared by using stereoselective oligonucleotide synthesis as described herein to form pre-designed internucleotide linkages that are diastereomerically pure with respect to a chiral linked phosphorus. Exemplary internucleotide linkages, including those having the structure of Formula I, are further described below.

[0395] In some embodiments, the present disclosure provides a chirally controlled oligonucleotide, wherein at least two individual internucleotide linkages within the oligonucleotide have different stereochemistry and / or different P modifications relative to each other.

[0396] Internucleotide linkage

[0397] In some embodiments, the provided oligonucleotides are capable of directing a decrease in the expression and / or level of a target gene or its gene product. In some embodiments, the provided oligonucleotides are capable of directing a decrease in the expression and / or level of a target gene or its gene product by RNA interference. In some embodiments, the provided oligonucleotides are capable of directing a decrease in the expression and / or level of a target gene or its gene product by a biochemical mechanism that does not involve RNA interference or RISC (including but not limited to RNase H-mediated knockdown or steric hindrance of gene expression). In some embodiments, the provided oligonucleotides are capable of directing a decrease in the expression and / or level of a target gene or its gene product by RNA interference and / or RNase H-mediated knockdown. In some embodiments, the provided oligonucleotides are capable of directing a decrease in the expression and / or level of a target gene or its gene product by annealing to the target gene mRNA and sterically blocking translation, and / or by altering or interfering with mRNA splicing and / or exon inclusion or exclusion. In some embodiments, the provided oligonucleotides comprise any internucleotide linkage described herein or known in the art.

[0398] In some embodiments, the APOC3 oligonucleotide, the APOC3 oligonucleotide that directs RNA interference, the APOC3 oligonucleotide that directs RNase H-mediated knockdown, or the APOC3 oligonucleotide that simultaneously directs RNA interference and RNase H-mediated knockdown may comprise any internucleotide linkage described herein or known in the art.

[0399] Non-limiting examples of internucleotide linkages or unmodified internucleotide linkages are phosphodiesters; non-limiting examples of modified internucleotide linkages include those internucleotide linkages in which one or more oxygens of the phosphodiester have been replaced by sulfur (such as the sulfur in phosphorothioates), H, alkyl, or another moiety or element that is not oxygen. Non-limiting examples of internucleotide linkages are moieties that do not contain phosphorus but are used to link two sugars. Non-limiting examples of internucleotide linkages are moieties that do not contain phosphorus but are used to link two sugars in the backbone of the APOC3 oligonucleotide. Other non-limiting examples of nucleotides, modified nucleotides, nucleotide analogs, internucleotide linkages, modified internucleotide linkages, bases, modified bases, and base analogs, sugars, modified sugars, and sugar analogs, and nucleosides, modified nucleosides, and nucleoside analogs are disclosed herein.

[0400] In certain embodiments, the internucleotide linkage has the structure of Formula I:

[0401]

[0402] where each variable is defined and described as follows. In some embodiments, the linkage of Formula I is chiral. In some embodiments, the present disclosure provides a chirally controlled oligonucleotide comprising one or more modified internucleotide linkages of Formula I. In some embodiments, the present disclosure provides a chirally controlled oligonucleotide comprising one or more modified internucleotide linkages of Formula I, and wherein the individual internucleotide linkages of Formula I within the oligonucleotide have different P modifications relative to one another. In some embodiments, the present disclosure provides a chirally controlled oligonucleotide comprising one or more modified internucleotide linkages of Formula I, and wherein the individual internucleotide linkages of Formula I within the oligonucleotide have different -X-L-R 1 . In some embodiments, the present disclosure provides a chirally controlled oligonucleotide comprising one or more modified internucleotide linkages of Formula I, and wherein the individual internucleotide linkages of Formula I within the oligonucleotide have different Xs relative to one another. In some embodiments, the present disclosure provides a chirally controlled oligonucleotide comprising one or more modified internucleotide linkages of Formula I, and wherein the individual internucleotide linkages of Formula I within the oligonucleotide have different -L-R relative to one another 1。In some embodiments, the chirally controlled oligonucleotide is an APOC3 oligonucleotide of a particular oligonucleotide type in the provided composition. In some embodiments, the chirally controlled oligonucleotide is an APOC3 oligonucleotide in the provided composition that has a common base sequence and length, a common backbone linkage pattern, and a common backbone chiral center pattern. In some embodiments, the chirally controlled oligonucleotide is an APOC3 oligonucleotide of a particular oligonucleotide type in a chirally controlled composition, and the chirally controlled oligonucleotide belongs to that type. In some embodiments, the chirally controlled oligonucleotide is an APOC3 oligonucleotide in the provided composition that comprises a plurality of oligonucleotides at a predetermined level, the plurality of oligonucleotides sharing a common base sequence, a common backbone linkage pattern, and a common backbone chiral center pattern, and the chirally controlled oligonucleotide sharing a common base sequence, a common backbone linkage pattern, and a common backbone chiral center pattern.

[0403] In some embodiments, the chirally controlled oligonucleotide comprises different internucleotide phosphorus linkages.

[0404] In some embodiments, the phosphorothioate triester linkage comprises a chiral auxiliary, such as for controlling the stereoselectivity of the reaction. In some embodiments, the phosphorothioate triester linkage does not comprise a chiral auxiliary. In some embodiments, the phosphorothioate triester linkage is intentionally maintained for administration to a subject and / or during administration to a subject.

[0405] In some embodiments, the chirally controlled oligonucleotide is attached to a solid support. In some embodiments, the chirally controlled oligonucleotide is cleaved from the solid support.

[0406] In some embodiments, the chirally controlled oligonucleotide comprises at least one phosphodiester internucleotide linkage and at least two consecutive modified internucleotide linkages. In some embodiments, the chirally controlled oligonucleotide comprises at least one phosphodiester internucleotide linkage and at least two consecutive phosphorothioate internucleotide linkages.

[0407] In some embodiments, the present disclosure provides a composition (e.g., a chirally controlled oligonucleotide composition) that comprises or consists of a plurality of the provided oligonucleotides. In some embodiments, all such provided oligonucleotides are of the same type, i.e., all have the same base sequence, backbone linkage pattern (i.e., internucleotide linkage type pattern, such as phosphate, phosphorothioate, etc.), backbone chiral center pattern (i.e., linkage phosphorus stereochemistry pattern (Rp / Sp)), and backbone phosphorus modification pattern (e.g., "-XLR" in Formula I disclosed herein 1”group pattern). In some embodiments, all oligonucleotides of the same type are identical. However, in many embodiments, the provided compositions typically contain a predetermined relative amount of multiple oligonucleotide types.

[0408] In some embodiments, the APOC3 oligonucleotide, the APOC3 oligonucleotide that directs RNA interference, the APOC3 oligonucleotide that directs RNase H-mediated knockdown, or the APOC3 oligonucleotide that simultaneously directs RNA interference and RNase H-mediated knockdown may contain any internucleotide linkage described herein or known in the art. In some embodiments, the moiety that binds to ASPGR is, for example, a GalNAc moiety, any GalNAc or its variants or modifications described herein or known in the art. In some embodiments, the APOC3 oligonucleotide, the APOC3 oligonucleotide that directs RNA interference, the APOC3 oligonucleotide that directs RNase H-mediated knockdown, or the APOC3 oligonucleotide that simultaneously directs RNA interference and RNase H-mediated knockdown may contain a combination of any internucleotide linkage described herein or known in the art with any other structural elements or modifications described herein, including but not limited to, base sequence, sugar, base (nucleobase) or portions thereof; stereochemistry or its pattern; additional chemical moieties, including but not limited to targeting moieties, lipid moieties, carbohydrate moieties, etc.; seed region; post-seed region; 5′-terminal structure; 5′-terminal region; 5′ nucleotide portion; 3′-terminal region; 3′-terminal dinucleotide; 3′-terminal cap; length; additional chemical moieties, including but not limited to targeting moieties, lipid moieties, GalNAc, etc.; its format or any structural element, and / or any other structural element or modification described herein; and in some embodiments, the present disclosure relates to a multimer of any such oligonucleotide.

[0409] In some embodiments, the chirality-controlled oligonucleotide contains one or more modified internucleotide phosphorus linkages. In some embodiments, the chirality-controlled oligonucleotide contains, for example, phosphorothioate or phosphorothioate triester linkages.

[0410] In some embodiments, the modified internucleotide linkage is a phosphorothioate. In some embodiments, the modified internucleotide linkage is selected from, for example, those described in: US 20110294124, US 20120316224, US20140194610, US 20150211006, US 20150197540, WO 2015107425, PCT / US2016 / 043542, and PCT / US2016 / 043598, Whittaker et al. 2008 Tetrahedron Letters 49: 6984-6987.

[0411] Non-limiting examples of internucleotide linkages also include those described in the art, including but not limited to those described in any of the following: Gryaznov, S.; Chen, J.-K. J. Am. Chem. Soc. 1994, 116, 3143, Jones et al. J. Org. Chem. 1993, 58, 2983, Koshkin et al. 1998 Tetrahedron 54: 3607-3630, Lauritsen et al. 2002 Chem. Comm. 5: 530-531, Lauritsen et al. 2003 Bioo. Med. Chem. Lett. 13: 253-256, Mesmaeker et al. Angew. Chem., Int. Ed. Engl. 1994, 33, 226, Petersen et al. 2003 TRENDS Biotech. 21: 74-81, Schultz et al. 1996 Nucleic Acids Res. 24: 2966, Ts′o et al. Ann. N.Y. Acad. Sci. 1988, 507, 220, and Vasseur et al. J. Am. Chem. Soc. 1992, 114, 4006.

[0412] In some embodiments, the present disclosure provides a chirally controlled oligonucleotide comprising a sequence present in any of the oligonucleotides disclosed herein. In some embodiments, the present disclosure provides a chirally controlled oligonucleotide comprising a sequence present in any of the oligonucleotides disclosed herein, wherein one or more U's are replaced by T's. In some embodiments, the present disclosure provides a chirally controlled oligonucleotide comprising a sequence present in any of the oligonucleotides disclosed herein, wherein the sequence has more than 50% identity to the sequence of any of the oligonucleotides disclosed herein.

[0413] In some embodiments, the present disclosure provides a chirally controlled oligonucleotide comprising a sequence of any of the oligonucleotides disclosed herein. In some embodiments, the present disclosure provides a chirally controlled oligonucleotide having a sequence of any of the oligonucleotides disclosed herein. In some embodiments, the present disclosure provides a chirally controlled oligonucleotide comprising a sequence present in any of the oligonucleotides disclosed herein, wherein the oligonucleotide has a backbone linkage pattern, a backbone chiral center pattern, and / or a backbone phosphorus modification pattern as described herein.

[0414] In some embodiments, the present disclosure provides a chirality-controlled oligonucleotide comprising a sequence present in any of the oligonucleotides disclosed herein. In some embodiments, the present disclosure provides a chirality-controlled oligonucleotide comprising a sequence present in any of the oligonucleotides disclosed herein, wherein one or more Ts are replaced by Us. In some embodiments, the present disclosure provides a chirality-controlled oligonucleotide comprising a sequence present in any of the oligonucleotides disclosed herein, wherein the sequence has more than 50% identity to the sequence of any of the oligonucleotides disclosed herein. In some embodiments, the present disclosure provides a chirality-controlled oligonucleotide comprising a sequence present in any of the oligonucleotides disclosed herein, wherein the sequence has more than 60% identity to the sequence of any of the oligonucleotides disclosed herein. In some embodiments, the present disclosure provides a chirality-controlled oligonucleotide comprising a sequence present in any of the oligonucleotides disclosed herein, wherein the sequence has more than 70% identity to the sequence of any of the oligonucleotides disclosed herein. In some embodiments, the present disclosure provides a chirality-controlled oligonucleotide comprising a sequence present in any of the oligonucleotides disclosed herein, wherein the sequence has more than 80% identity to the sequence of any of the oligonucleotides disclosed herein. In some embodiments, the present disclosure provides a chirality-controlled oligonucleotide comprising a sequence present in any of the oligonucleotides disclosed herein, wherein the sequence has more than 90% identity to the sequence of any of the oligonucleotides disclosed herein. In some embodiments, the present disclosure provides a chirality-controlled oligonucleotide comprising a sequence present in any of the oligonucleotides disclosed herein, wherein the sequence has more than 95% identity to the sequence of any of the oligonucleotides disclosed herein. In some embodiments, the present disclosure provides a chirality-controlled oligonucleotide comprising a sequence of any of the oligonucleotides disclosed herein. In some embodiments, the present disclosure provides a chirality-controlled oligonucleotide having a sequence of any of the oligonucleotides disclosed herein.

[0415] The present disclosure provides a chirality-controlled oligonucleotide comprising a sequence of any of the oligonucleotides disclosed herein, wherein at least one internucleotide linkage is The present disclosure provides a chirality-controlled oligonucleotide comprising a sequence of any of the oligonucleotides disclosed herein, wherein each internucleotide linkage is The present disclosure provides a chirality-controlled oligonucleotide having a sequence of any of the oligonucleotides disclosed herein, wherein at least one internucleotide linkage is The present disclosure provides a chirality-controlled oligonucleotide having a sequence of any of the oligonucleotides disclosed herein, wherein each internucleotide linkage is In some embodiments, the present disclosure provides a chirality-controlled oligonucleotide having the sequence of any oligonucleotide disclosed herein, wherein each cytosine is optionally and independently replaced by 5-methylcytosine. In some embodiments, the present disclosure provides a chirality-controlled oligonucleotide having the sequence of any oligonucleotide disclosed herein, wherein at least one cytosine is optionally and independently replaced by 5-methylcytosine. In some embodiments, the present disclosure provides a chirality-controlled oligonucleotide having the sequence of any oligonucleotide disclosed herein, wherein each cytosine is optionally and independently replaced by 5-methylcytosine.

[0416] Base (nucleobase)

[0417] In some embodiments, the provided oligonucleotide is capable of directing a decrease in the expression and / or level of a target gene or its gene product. In some embodiments, the provided oligonucleotide is capable of directing a decrease in the expression and / or level of a target gene or its gene product through RNA interference. In some embodiments, the provided oligonucleotide is capable of directing a decrease in the expression and / or level of a target gene or its gene product through a biochemical mechanism that does not involve RNA interference or RISC (including but not limited to RNase H-mediated knockdown or steric hindrance of gene expression). In some embodiments, the provided oligonucleotide is capable of directing a decrease in the expression and / or level of a target gene or its gene product through RNA interference and / or RNA

[0418] H-mediated knockdown. In some embodiments, the provided oligonucleotide is capable of directing a decrease in the expression and / or level of a target gene or its gene product by annealing to the target gene mRNA and sterically blocking translation, and / or by altering or interfering with mRNA splicing and / or exon inclusion or exclusion. In some embodiments, the provided oligonucleotide comprises any nucleobase described herein or known in the art.

[0419] In some embodiments, the nucleobases present in the provided oligonucleotides are natural nucleobases or modified nucleobases derived from natural nucleobases. Examples include, but are not limited to, uracil, thymine, adenine, cytosine, and guanine in which the corresponding amino group is protected by an acyl protecting group; 2-fluorouracil, 2-fluorocytosine, 5-bromouracil, 5-iodouracil, 2,6-diaminopurine, azacytosine, pyrimidine analogs (such as pseudoisocytosine and pseudouracil), and other modified nucleobases such as 8-substituted purines, xanthine, or hypoxanthine (the latter two being natural degradation products). Exemplary modified nucleobases are disclosed in Chiu and Rana, RNA, 2003, 9, 1034-1048, Limbach et al. Nucleic Acids Research, 1994, 22, 2183-2196, and Revankar and Rao, Comprehensive Natural Products Chemistry, Volume 7, 313. In some embodiments, the modified nucleobases are substituted uracil, thymine, adenine, cytosine, or guanine. In some embodiments, the modified nucleobases are functional replacements of uracil, thymine, adenine, cytosine, or guanine, for example, with respect to hydrogen bonding and / or base pairing. In some embodiments, the nucleobases are optionally substituted uracil, thymine, adenine, cytosine, 5-methylcytosine, or guanine. In some embodiments, the nucleobases are uracil, thymine, adenine, cytosine, 5-methylcytosine, or guanine.

[0420] In some embodiments, the modified bases are optionally substituted adenine, cytosine, guanine, thymine, or uracil. In some embodiments, the modified nucleobases are independently adenine, cytosine, guanine, thymine, or uracil, which are modified by one or more modifications by which:

[0421] (1) the nucleobase is modified by one or more optionally substituted groups independently selected from: acyl, halogen, amino, azide, alkyl, alkenyl, alkynyl, aryl, heteroalkyl, heteroalkenyl, heteroalkynyl, heterocyclic, heteroaryl, carboxyl, hydroxyl, biotin, avidin, streptavidin, substituted silyl, and combinations thereof;

[0422] (2) one or more atoms of the nucleobase are independently replaced by different atoms selected from carbon, nitrogen, and sulfur;

[0423] (3) one or more double bonds in the nucleobase are independently hydrogenated; or

[0424] (4) one or more aryl or heteroaryl rings are independently inserted into the nucleobase.

[0425] A variety of additional nucleobases are described in the art. Sugar

[0426] In some embodiments, the provided oligonucleotides are capable of directing a reduction in the expression and / or level of a target gene or its gene product. In some embodiments, the provided oligonucleotides are capable of directing a reduction in the expression and / or level of a target gene or its gene product through RNA interference. In some embodiments, the provided oligonucleotides are capable of directing a reduction in the expression and / or level of a target gene or its gene product through biochemical mechanisms that do not involve RNA interference or RISC (including but not limited to RNase H-mediated knockdown or steric hindrance of gene expression). In some embodiments, the provided oligonucleotides are capable of directing a reduction in the expression and / or level of a target gene or its gene product through RNA interference and / or RNase H-mediated knockdown. In some embodiments, the provided oligonucleotides are capable of directing a reduction in the expression and / or level of a target gene or its gene product by sterically blocking translation upon annealing to the target gene mRNA, and / or by altering or interfering with mRNA splicing and / or exon inclusion or exclusion. In some embodiments, the provided oligonucleotides comprise any sugar described herein or known in the art.

[0427] In some embodiments, the provided oligonucleotides capable of directing single-stranded RNA interference comprise one or more modified sugar moieties in addition to the native sugar moiety.

[0428] The most common naturally occurring nucleotides comprise ribose linked to the nucleobases adenosine (A), cytosine (C), guanine (G), and thymine (T) or uracil (U). Modified nucleotides are also encompassed, where the phosphate ester group or the linked phosphorus in the nucleotide can be attached to various positions of the sugar or modified sugar. As non-limiting examples, the phosphate ester group or the linked phosphorus can be attached to the 2″, 3″, 4″, or 5″ hydroxyl moiety of the sugar or modified sugar. In this context, nucleotides incorporating modified nucleobases as described herein are also encompassed. In some embodiments, nucleotides or modified nucleotides comprising unprotected -OH moieties are used in the methods according to the present disclosure.

[0429] In some embodiments, an APOC3 oligonucleotide, an APOC3 oligonucleotide that directs RNA interference, an APOC3 oligonucleotide that directs RNase H-mediated knockdown, or an APOC3 oligonucleotide that simultaneously directs RNA interference and RNase H-mediated knockdown may comprise any base (nucleobase), modified base, or base analog described herein or known in the art. In some embodiments, an APOC3 oligonucleotide, an APOC3 oligonucleotide that directs RNA interference, an APOC3 oligonucleotide that directs RNase H-mediated knockdown, or an APOC3 oligonucleotide that simultaneously directs RNA interference and RNase H-mediated knockdown may comprise any combination of any base described herein or known in the art with any other structural element or modification described herein, including but not limited to, a base sequence or a portion thereof; a sugar; an internucleotide linkage; stereochemistry or a pattern thereof; additional chemical moieties, including but not limited to a targeting moiety, a lipid moiety, a GalNAc moiety, etc.; a 5'-terminal structure; a 5'-terminal region; a 5'-nucleotide portion; a seed region; a post-seed region; a 3'-terminal region; a 3'-terminal dinucleotide; a 3'-terminal cap; a modified pattern of a sugar, a base, or an internucleotide linkage; its format or any structural element, and / or any other structural element or modification described herein; and in some embodiments, the present disclosure relates to a multimer of any such oligonucleotide.

[0430] In some embodiments, an APOC3 oligonucleotide, an APOC3 oligonucleotide that directs RNA interference, an APOC3 oligonucleotide that directs RNase H-mediated knockdown, or an APOC3 oligonucleotide that simultaneously directs RNA interference and RNase H-mediated knockdown may comprise any sugar.

[0431] A variety of additional sugars have been described in the art.

[0432] The base sequence of the APOC3 oligonucleotide

[0433] In some embodiments, the provided oligonucleotides are capable of directing a reduction in the expression and / or level of a target gene or its gene product. In some embodiments, the provided oligonucleotides are capable of directing a reduction in the expression and / or level of a target gene or its gene product through RNA interference. In some embodiments, the provided oligonucleotides are capable of directing a reduction in the expression and / or level of a target gene or its gene product through a biochemical mechanism that does not involve RNA interference or RISC (including but not limited to RNase H-mediated knockdown or steric hindrance of gene expression). In some embodiments, the provided oligonucleotides are capable of directing a reduction in the expression and / or level of a target gene or its gene product through RNA interference and / or RNase H-mediated knockdown. In some embodiments, the provided oligonucleotides are capable of directing a reduction in the expression and / or level of a target gene or its gene product by annealing to the target gene mRNA and sterically blocking translation spatially, and / or by altering or interfering with mRNA splicing and / or exon inclusion or exclusion. In some embodiments, the provided oligonucleotides may comprise any of the base sequences described herein or portions thereof, wherein a portion is a span of at least 15 contiguous bases or a span of at least 15 contiguous bases having 1-5 mismatches.

[0434] In some embodiments, an APOC3 oligonucleotide, an APOC3 oligonucleotide that directs RNA interference, an APOC3 oligonucleotide that directs RNase H-mediated knockdown, or an APOC3 oligonucleotide that simultaneously directs RNA interference and RNase H-mediated knockdown may comprise any of the base sequences described herein. In some embodiments, an APOC3 oligonucleotide, an APOC3 oligonucleotide that directs RNA interference, an APOC3 oligonucleotide that directs RNase H-mediated knockdown, or an APOC3 oligonucleotide that simultaneously directs RNA interference and RNase H-mediated knockdown may comprise any of the base sequences described herein or portions thereof. In some embodiments, an APOC3 oligonucleotide, an APOC3 oligonucleotide that directs RNA interference, an APOC3 oligonucleotide that directs RNase H-mediated knockdown, or an APOC3 oligonucleotide that simultaneously directs RNA interference and RNase H-mediated knockdown may comprise any of the base sequences described herein or portions thereof, wherein a portion is a span of 15 contiguous bases or a span of 15 contiguous bases having 1-5 mismatches.

[0435] The sequence of a single-stranded RNAi agent has sufficient length and identity to the transcript target to mediate target-specific RNA interference. In some embodiments, the RNAi agent is complementary to a portion of the transcript target sequence.

[0436] The base sequence of the single-stranded RNAi agent is complementary to the base sequence of the target transcript. As used herein, "target transcript sequence", "target sequence", "target gene", etc. refer to a continuous portion of the nucleotide sequence of an mRNA molecule formed during gene transcription, said gene being, for example, a target gene, including the mRNA that is the product of RNA processing of the primary transcript.

[0437] The terms "complementary", "fully complementary", and "substantially complementary" can be used herein with respect to base pairing between the strand of a single-stranded RNAi agent and the target sequence or between an antisense oligonucleotide and the target sequence, as can be understood from the context in which they are used. A strand of a single-stranded RNAi agent, antisense oligonucleotide, or other oligonucleotide is complementary to a strand of the target sequence when each base of the single-stranded RNAi agent, antisense oligonucleotide, or other oligonucleotide is capable of base pairing with the sequential bases on the target strand, when maximally aligned. As a non-limiting example, if the target sequence has a base sequence such as 5′-GCAUAGCGAGCGAGGGAAAAC-3′, an APOC3 oligonucleotide having the base sequence 5′GUUUUCCCUCGCUCGCUAUGC-3′ is complementary or fully complementary to such a target sequence. Of course, it should be noted that replacing U with T or vice versa does not change the amount of complementarity.

[0438] As used herein, a polynucleotide that is "substantially complementary" to a target sequence is complementary to a large extent or for the most part, but not 100% complementary. In some embodiments, a substantially complementary sequence (e.g., a strand of a single-stranded RNAi agent or an antisense oligonucleotide) has 1, 2, 3, 4, or 5 mismatches with a sequence that is 100% complementary to the target sequence. In the case of a single-stranded RNAi agent, the present disclosure indicates that in many cases the 5′-terminal nucleotide (N1) has a mismatch with the complementary sequence of the target sequence. Similarly, in a single-stranded RNAi agent, the 3′-terminal dinucleotide, if present, can have a mismatch with the complementary sequence of the target sequence. As a non-limiting example, if the target sequence has a base sequence such as 5′-GCAUAGCGAGCGAGGGAAAAC-3′, a single-stranded RNAi agent having the base sequence 5′TUUUUCCCUCGCUCGCUAUTU-3′ is substantially complementary to such a target sequence.

[0439] The present disclosure presents various single-stranded RNAi agents, antisense oligonucleotides, and other oligonucleotides in Table 1A and elsewhere, each of which has a defined base sequence. In some embodiments, the present disclosure encompasses any oligonucleotide having a base sequence that is the same as, includes, or includes a portion of: any of the various single

[0440] Base sequences of RNAi agents, antisense oligonucleotides, and other oligonucleotides. In some embodiments, the present disclosure encompasses any oligonucleotide having a base sequence that is, consists of, or consists of a portion of: the base sequences of any of the various single-stranded RNAi agents, antisense oligonucleotides, and other oligonucleotides disclosed herein, which have any chemical modification, stereochemistry, format, structural feature (e.g., if the oligonucleotide is a single

[0441] stranded RNAi agent, the 5′-terminal structure, 5′-terminal region, 5′ nucleotide portion, seed region, post-seed region, 3′-terminal region, 3′-terminal dinucleotide, 3′-terminal cap, or any structure, pattern, or portion thereof) and / or any other modification described herein (e.g., conjugation to another moiety such as a targeting moiety, carbohydrate moiety, GalNAc moiety, lipid moiety, etc.; and / or polymerization).

[0442] In some embodiments, the base sequence of the APOC3 oligonucleotide is, consists of, or consists of a portion of: the base sequence of any oligonucleotide disclosed herein.

[0443] In some embodiments, the present disclosure discloses APOC3 oligonucleotides of the sequences described herein. In some embodiments, the present disclosure discloses APOC3 oligonucleotides of the sequences described herein, wherein the oligonucleotides are capable of directing a reduction in the expression and / or level of a target gene or its gene product. In some embodiments, the APOC3 oligonucleotides of the sequences are single-stranded RNAi agents. In some embodiments, the APOC3 oligonucleotides of the sequences are antisense oligonucleotides that direct RNase H-mediated knockdown. In some embodiments, the APOC3 oligonucleotides of the sequences direct both RNA interference and RNase H-mediated knockdown simultaneously. In some embodiments, the oligAPOC3 nucleotides of the sequences comprise any of the structures described herein (e.g., any 5′-terminal structure, 5′-terminal region, 5′ nucleotide portion, seed region, post-seed region, 3′-terminal dinucleotide, 3′-terminal cap, or any portion of any of these structures, or any chemical, stereochemical, additional chemical moiety, etc.). If the oligonucleotide is an ssRNAi agent, it may be preceded by a T (as non-limiting examples, 2′-deoxy T, 5′-(R)-Me OH T, 5′-(R)-Me PO T, 5′-(R)-Me PS T, 5′-(R)-Me PH T, 5′-(S)-Me OH T, 5′-(S)-Me PO T, 5′-(S)-Me PS T, or 5′-(S)-PH T) or the first nucleobase may be replaced by a T (as non-limiting examples, 2′-deoxy T, 5′-(R)-Me OH T, 5′-(R)-Me PO T, 5′-(R)-Me PS T, 5′-(R)-Me PH T, 5′-(S)-Me OH T, 5′-(S)-Me PO T, 5′-(S)-Me PS T, or 5′-(S)-PH T), and / or may be followed by a 3′-terminal dinucleotide (e.g., as non-limiting examples: TT, UU, TU, etc.). In various sequences, U may be replaced by T, or vice versa, or the sequence may contain a mixture of U and T. In some embodiments, the APOC3 oligonucleotides are no more than about 49, 45, 40, 30, 35, 25, 23 nucleotides in length in total. In some embodiments, the portion is a span of at least 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides in total with 0-3 mismatches. In some embodiments, the portion is a span of at least 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides in total with 0-3 mismatches, wherein the span with 0 mismatches is complementary and the spans with 1 or more mismatches are non-limiting examples of sequences that are substantially complementary.In some embodiments, where the above sequence starts with a U at the 5′-end, the U can be absent and / or replaced by another base. In some embodiments, the present disclosure encompasses any oligonucleotide having a base sequence that is the following or that comprises the following or that comprises a portion of the following: any oligonucleotide disclosed herein having a format or a portion of a format disclosed herein.

[0444] In some embodiments, an APOC3 oligonucleotide, an APOC3 oligonucleotide that directs RNA interference, an APOC3 oligonucleotide that directs RNase H-mediated knockdown, or an APOC3 oligonucleotide that simultaneously directs RNA interference and RNase H-mediated knockdown can comprise any base sequence described herein. In some embodiments, an APOC3 oligonucleotide, an APOC3 oligonucleotide that directs RNA interference, an APOC3 oligonucleotide that directs RNase H-mediated knockdown, or an APOC3 oligonucleotide that simultaneously directs RNA interference and RNase H-mediated knockdown can comprise any base sequence described herein or a portion thereof. In some embodiments, an APOC3 oligonucleotide, an APOC3 oligonucleotide that directs RNA interference, an APOC3 oligonucleotide that directs RNase H-mediated knockdown, or an APOC3 oligonucleotide that simultaneously directs RNA interference and RNase H-mediated knockdown can comprise any base sequence described herein or a portion thereof, where the portion is a span of 15 consecutive bases or a span of 15 consecutive bases having 1-5 mismatches. In some embodiments, an APOC3 oligonucleotide, an APOC3 oligonucleotide that directs RNA interference, an APOC3 oligonucleotide that directs RNase H-mediated knockdown, or an APOC3 oligonucleotide that simultaneously directs RNA interference and RNase H-mediated knockdown can comprise any base sequence described herein or a portion thereof in combination with any other structural element or modification described herein, including but not limited to, a sugar; a base; an internucleotide linkage; a stereochemistry or a pattern thereof; an additional chemical moiety, including but not limited to, a targeting moiety, a lipid moiety, a GalNAc moiety, etc.; a 5′-end structure; a 5′-end region; a 5′ nucleotide portion; a seed region; a post-seed region; a 3′-end region; a 3′-end dinucleotide; a 3′-end cap; a modified pattern of a sugar, a base, or an internucleotide linkage; a format or any structural element thereof, and / or any other structural element or modification described herein; and in some embodiments, the present disclosure relates to a multimer of any such oligonucleotide.

[0445] Non-limiting examples of oligonucleotides having various base sequences are disclosed in Table 1A below.

[0446]

[0447]

[0448]

[0449]

[0450]

[0451]

[0452]

[0453]

[0454]

[0455]

[0456]

[0457]

[0458]

[0459]

[0460]

[0461]

[0462]

[0463]

[0464]

[0465]

[0466]

[0467]

[0468]

[0469]

[0470]

[0471]

[0472]

[0473]

[0474]

[0475]

[0476]

[0477]

[0478]

[0479]

[0480]

[0481]

[0482]

[0483]

[0484]

[0485]

[0486]

[0487]

[0488]

[0489]

[0490]

[0491]

[0492]

[0493]

[0494]

[0495]

[0496]

[0497]

[0498]

[0499]

[0500]

[0501]

[0502]

[0503]

[0504]

[0505]

[0506]

[0507]

[0508]

[0509]

[0510]

[0511]

[0512]

[0513]

[0514]

[0515]

[0516]

[0517]

[0518]

[0519]

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[0631] In some embodiments, the provided oligonucleotide compositions are single-stranded RNAi agents listed in Table 1A or described elsewhere herein. In some embodiments, exemplary properties of the provided oligonucleotides are demonstrated.

[0632] In some embodiments, the provided oligonucleotides have a structure in any of the formats shown in FIG. 1.

[0633] The present disclosure presents numerous non-limiting examples of oligonucleotides (e.g., single-stranded RNAi agents) that are capable of mediating single-stranded RNA interference. Experimental data (not shown) indicate that various putative single-stranded RNAi agents are indeed capable of mediating RNA interference. In some experiments, an in vitro Ago-2 assay was used, including the use of the RNA test substrate WV-2372 (APOC3). In the presence of the oligonucleotides WV-1308 and WV-2420, there was no band representing the RNA test substrate, indicating that these oligonucleotides are single-stranded RNAi agents capable of mediating RNA interference. The remaining lanes were controls: substrate without the negative control ASO WV-2134; substrate without the negative control ASO WV-2134 that does not mediate RNA interference; substrate without the test oligonucleotide WV-1308; substrate without the test oligonucleotide WV-2420; substrate only; no substrate with WV-2134 added; and no substrate with WV-1308 added. An in vitro Ago-2 assay was also performed (data not shown) that used APOC3 mRNA as the test substrate in a Hep3B cell 3' RACE assay. In the presence of the test oligonucleotide WV-3021, a cleavage product of APOC3 mRNA was detected, which corresponded to the cleavage of the mRNA at the site of a cleavage between positions 10 and 11 corresponding to WV-3021. An artificial cleavage product was also detected. Experimental data (not shown) demonstrated that the dual-mechanism oligonucleotide WV-2111 was capable of mediating knockdown simultaneously by RNase H and RNA interference. In the experiment, several oligonucleotides were capable of mediating RNA interference. The RNA test substrate was WV-2372. The experiment showed that in the presence of the test oligonucleotides WV-1308, WV-2114, WV-2386, and WV-2387, the RNA test substrate disappeared, indicating that all of these oligonucleotides were capable of acting as single-stranded RNAi agents mediating RNA interference. The remaining lanes were controls. Thus, the experiment showed that the oligonucleotides WV-1308, WV-2114, WV-2386, and WV-2387 were all capable of mediating RNA interference. Thus, the experiment showed that several single-stranded RNAi agents (e.g., WV-1308, WV-2420, WV-3021, WV-2111, WV-2114, WV-2386, and WV-2387) were capable of mediating RNA interference. The present disclosure presents numerous non-limiting examples of oligonucleotides that have any of various sequences, formats, modifications, 5'-terminal regions, seed regions, post-seed regions, and 3'-terminal regions and are capable of mediating single-stranded RNA interference (e.g., single-stranded RNAi agents).

[0634] Format of the oligonucleotide

[0635] In some embodiments, the provided oligonucleotides are capable of directing a decrease in the expression and / or level of a target gene or its gene product. In some embodiments, the provided oligonucleotides are capable of directing a decrease in the expression and / or level of a target gene or its gene product through RNA interference. In some embodiments, the provided oligonucleotides are capable of directing a decrease in the expression and / or level of a target gene or its gene product through biochemical mechanisms that do not involve RNA interference or RISC (including but not limited to RNase H-mediated knockdown or steric hindrance of gene expression). In some embodiments, the provided oligonucleotides are capable of directing a decrease in the expression and / or level of a target gene or its gene product through RNA interference and / or RNAH-mediated knockdown. In some embodiments, the provided oligonucleotides are capable of directing a decrease in the expression and / or level of a target gene or its gene product by spatially blocking translation after annealing of the target gene mRNA and / or by altering or interfering with mRNA splicing and / or exon inclusion or exclusion. In some embodiments, the provided oligonucleotides may have any format or portion or structural element thereof described herein or known in the art.

[0636] In some embodiments, the APOC3 oligonucleotides may have any format or structural element thereof described herein or known in the art.

[0637] In some embodiments, the APOC3 oligonucleotides capable of directing a decrease in the expression and / or level of a target gene or its gene product may have any format or structural element thereof described herein or known in the art.

[0638] In some embodiments, the APOC3 oligonucleotides, the APOC3 oligonucleotides that direct RNA interference, the APOC3 oligonucleotides that direct RNase H-mediated knockdown, or the APOC3 oligonucleotides that direct both RNA interference and RNase H-mediated knockdown may have any format or structural element thereof described herein or known in the art.

[0639] Additional non-limiting examples of various ssRNAi formats are exemplified by the various single-stranded RNAi agents described herein.

[0640] In some embodiments, the provided single-stranded RNAi comprises a 5'-end represented by any single-stranded RNAi format depicted in FIG. 1 or any single-stranded RNAi agent or any 5'-end of a single-stranded RNAi format described herein.

[0641] In some embodiments, the provided single-stranded RNAi comprises a 5'-end structure or 5'-end region represented by any single-stranded RNAi format depicted in FIG. 1 or any single-stranded RNAi agent or any 5'-end structure or 5'-end region of a single-stranded RNAi format described herein.

[0642] In some embodiments, the provided single-stranded RNAi comprises a 5'-nucleotide represented by any of the single-stranded RNAi formats depicted in FIG. 1 or any 5'-nucleotide of any single-stranded RNAi agent or single-stranded RNAi format described herein.

[0643] In some embodiments, the provided single-stranded RNAi comprises a 5'-nucleoside represented by any of the single-stranded RNAi formats depicted in FIG. 1 or any 5'-nucleoside of any single-stranded RNAi agent or single-stranded RNAi format described herein.

[0644] In some embodiments, the provided single-stranded RNAi comprises a seed region represented by any of the single-stranded RNAi formats depicted in FIG. 1 or any seed region of any single-stranded RNAi agent or single-stranded RNAi format described herein.

[0645] In some embodiments, the provided single-stranded RNAi comprises a post-seed region represented by any of the single-stranded RNAi formats depicted in FIG. 1 or any post-seed region of any single-stranded RNAi agent or single-stranded RNAi format described herein.

[0646] In some embodiments, the provided single-stranded RNAi comprises a post-seed region or a component thereof represented by any of the single-stranded RNAi formats depicted in FIG. 1 or any post-seed region or a component thereof of any single-stranded RNAi agent or single-stranded RNAi format described herein.

[0647] In some embodiments, the provided single-stranded RNAi comprises a 3'-terminal dinucleotide represented by any of the single-stranded RNAi formats depicted in FIG. 1 or any 3'-terminal dinucleotide of any single-stranded RNAi agent or single-stranded RNAi format described herein.

[0648] In some embodiments, the provided single-stranded RNAi comprises a seed region having a nucleotide internucleoside linkage pattern represented by any of the single-stranded RNAi formats depicted in FIG. 1 or any nucleotide internucleoside linkage pattern of any seed region of any single-stranded RNAi agent or single-stranded RNAi format described herein.

[0649] In some embodiments, the provided single-stranded RNAi comprises a post-seed region having a nucleotide internucleoside linkage pattern represented by any of the single-stranded RNAi formats depicted in FIG. 1 or any nucleotide internucleoside linkage pattern of any post-seed region of any single-stranded RNAi agent or single-stranded RNAi format described herein.

[0650] In some embodiments, the provided single-stranded RNAi comprises a post-seed region or a component thereof having an internucleotide linkage pattern represented by the internucleotide linkage pattern of any post-seed region or a component thereof of any single-stranded RNAi format depicted in FIG. 1 or any single-stranded RNAi agent or single-stranded RNAi format described herein.

[0651] In some embodiments, the provided single-stranded RNAi comprises a 3'-terminal dinucleotide having an internucleotide linkage pattern represented by the internucleotide linkage pattern of any 3'-terminal dinucleotide of any single-stranded RNAi format depicted in FIG. 1 or any single-stranded RNAi agent or single-stranded RNAi format described herein.

[0652] In some embodiments, the provided single-stranded RNAi comprises a seed region having a chemical modification pattern represented by the chemical modification pattern of any seed region of any single-stranded RNAi format depicted in FIG. 1 or any single-stranded RNAi agent or single-stranded RNAi format described herein.

[0653] In some embodiments, the provided single-stranded RNAi comprises a post-seed region having a chemical modification pattern represented by the chemical modification pattern of any post-seed region of any single-stranded RNAi format depicted in FIG. 1 or any single-stranded RNAi agent or single-stranded RNAi format described herein.

[0654] In some embodiments, the provided single-stranded RNAi comprises a post-seed region or a component thereof having a chemical modification pattern represented by the chemical modification pattern of any post-seed region or a component thereof of any single-stranded RNAi format depicted in FIG. 1 or any single-stranded RNAi agent or single-stranded RNAi format described herein.

[0655] In some embodiments, the provided single-stranded RNAi comprises a 3'-terminal dinucleotide having a chemical modification pattern represented by the chemical modification pattern of any 3'-terminal dinucleotide of any single-stranded RNAi format depicted in FIG. 1 or any single-stranded RNAi agent or single-stranded RNAi format described herein.

[0656] In some embodiments, the provided single-stranded RNAi comprises a chemical modification represented by any chemical modification of any single-stranded RNAi format depicted in FIG. 1 or any single-stranded RNAi agent or single-stranded RNAi format described herein.

[0657] In some embodiments, the provided single-stranded RNAi comprises a chemical modification represented by any chemical modification of any single-stranded RNAi format depicted in FIG. 1 or described herein, wherein the chemical modification is a conjugation of a moiety comprising a phosphate ester, a linker, or a targeting moiety.

[0658] In some embodiments, the provided single-stranded RNAi comprises chemical modifications represented by any of the single-stranded RNAi formats depicted in Figure 1 or described herein, wherein the chemical modification is a conjugation of a moiety comprising a phosphate, a linker, or a targeting moiety, and wherein the targeting moiety comprises a GalNAc moiety. In some embodiments, the GalNAc is protected or deprotected GalNAc.

[0659] In some embodiments, the APOC3 oligonucleotide is capable of reducing the expression, activity, and / or level of a target gene and / or its gene product and has the format of any oligonucleotide described herein. In some embodiments, the APOC3 oligonucleotide is capable of reducing the expression, activity, and / or level of a target gene and / or its gene product by an RNase H-mediated mechanism or a mechanism related to steric hindrance of translation and has the format of any oligonucleotide described herein. In some embodiments, the APOC3 oligonucleotide is capable of reducing the expression, activity, and / or level of a target gene and / or its gene product by an RNase H-mediated mechanism or a mechanism related to steric hindrance of translation and has an asymmetric format. In some embodiments, the APOC3 oligonucleotide having an asymmetric format comprises a first wing, a core, and a second wing, wherein the core comprises a region of 5 or more contiguous 2'-deoxynucleotides that can anneal to the target mRNA and form a structure recognized by RNase H, and wherein the structures of the first wing and the second wing are different. In some embodiments, the first wing and the second wing are different in terms of their 2'-modifications and / or the stereochemical pattern of the internucleotide linkage or internucleotide linkages.

[0660] In some embodiments, the APOC3 oligonucleotide is capable of reducing the expression, activity, and / or level of a target gene and / or its gene product and comprises a neutral internucleotide linkage (e.g., a neutral backbone).

[0661] In some embodiments, the APOC3 oligonucleotide comprises a neutral backbone. In some embodiments, the APOC3 oligonucleotide comprises an internucleotide linkage that is a triazole, a neutral triazole, or an alkyne. In some embodiments, a nucleic acid (including but not limited to an APOC3 oligonucleotide) comprises an internucleotide linkage that comprises a triazole, a neutral triazole, or an alkyne, wherein the internucleotide linkage is stereocontrolled and has an Rp or Sp configuration. In some embodiments, the internucleotide linkage comprising a triazole has the following formula: In some embodiments, the internucleotide linkage comprising a neutral triazole has the following formula:

[0662] wherein X is O or S. In some embodiments, the internucleotide linkage comprising an alkyne has the following formula: wherein X is O or S.

[0663] In some embodiments, the internucleotide linkage comprises a cyclic guanidine. In some embodiments, the internucleotide linkage comprises a cyclic guanidine moiety and has the following structure: In some embodiments, the neutral internucleotide linkage or internucleotide linkage comprising a cyclic guanidine is stereochemically controlled. In some embodiments, the neutral internucleotide linkage improves the activity, delivery, and / or stability of the APOC3 oligonucleotide and / or the ability of the APOC3 oligonucleotide to effect endosomal escape.

[0664] As will be appreciated by those skilled in the art, in some cases, can be used to indicate a linkage site (such as ); in some cases, can be used to indicate a stereorandom linkage.

[0665] The length of the APOC3 oligonucleotide

[0666] In some embodiments, the provided oligonucleotide is capable of directing a decrease in the expression and / or level of a target gene or its gene product. In some embodiments, the provided oligonucleotide is capable of directing a decrease in the expression and / or level of a target gene or its gene product by RNA interference. In some embodiments, the provided oligonucleotide is capable of directing a decrease in the expression and / or level of a target gene or its gene product by a biochemical mechanism that does not involve RNA interference or RISC (including but not limited to RNase H-mediated knockdown or steric hindrance of gene expression). In some embodiments, the provided oligonucleotide is capable of directing a decrease in the expression and / or level of a target gene or its gene product by RNA interference and / or RNase H-mediated knockdown. In some embodiments, the provided oligonucleotide is capable of directing a decrease in the expression and / or level of a target gene or its gene product by annealing to the target gene mRNA and sterically blocking translation, and / or by altering or interfering with mRNA splicing and / or exon inclusion or exclusion. In some embodiments, the provided oligonucleotide can have any length, wherein the length of the APOC3 oligonucleotide is such that the oligonucleotide is capable of directing a decrease in the expression and / or level of a target gene or its gene product.

[0667] In some embodiments, the length of the APOC3 oligonucleotide is such that the oligonucleotide is capable of directing a decrease in the expression and / or level of a target gene or its gene product.

[0668] In some embodiments, the length of the RNAi agent is such that the RNAi agent is capable of directing RNA interference of a specific target transcript in a sequence-specific manner. In some embodiments, the RNAi agent comprises a sufficient number of nucleobases with sufficient identity to recognize the target transcript. In some embodiments, the RNAi agent also has a length suitable for mediating RNA interference.

[0669] The target portion of the sequence will be at least long enough to serve as a substrate for iRNA-directed cleavage at or near that portion. For example, the length of the target sequence is typically about 9 - 36 nucleotides ("nt"), such as about 15 - 30 nucleotides in length, including all sub-ranges therebetween. Table 1A shows examples of single-stranded RNAi agents of various lengths.

[0670] Figure 1 shows non-limiting examples of single-stranded RNAi agents that are 19 to 25 in length. Single-stranded RNAi agents were constructed with each of these lengths, and they were found to be capable of knocking down the target gene. Thus, the provided single-stranded RNAi agents can have any of a number of different lengths.

[0671] Non-limiting examples of the format of the ssRNAi agent that is 19 bases in length include: Format 20 - 21 of Figure 1.

[0672] Non-limiting examples of the format of the ssRNAi agent that is 20 bases in length include: Format 19 of Figure 1.

[0673] The 5'-end of the APOC3 oligonucleotide comprising the single-stranded RNAi agent

[0674] In some embodiments, the structure of the 5'-end of the oligonucleotide is such that the APOC3 oligonucleotide is capable of directing a decrease in the expression and / or level of the target gene or its gene product.

[0675] In some embodiments, the structure of the 5'-end of the RNAi agent is such that the RNAi agent is capable of directing RNA interference of a specific target transcript in a sequence-specific manner.

[0676] In some embodiments, the provided oligonucleotides can comprise any 5'-terminal region, 5'-terminal structure, 5'-terminal group, 5'-terminal nucleoside, or 5'-terminal nucleotide described herein or known in the art. In some embodiments, the provided oligonucleotides capable of directing RNase H-mediated knockdown can comprise any 5'-terminal region, 5'-terminal structure, 5'-terminal group, 5'-terminal nucleoside, or 5'-terminal nucleotide described herein or known in the art. In some embodiments, the provided oligonucleotides capable of directing RNA interference can comprise any 5'-terminal region, 5'-terminal structure, 5'-terminal group, 5'-terminal nucleoside, or 5'-terminal nucleotide described herein or known in the art. In some embodiments, the provided oligonucleotides capable of directing both RNA interference and RNase H-mediated knockdown can comprise any 5'-terminal region, 5'-terminal structure, 5'-terminal group, 5'-terminal nucleoside, or 5'-terminal nucleotide described herein or known in the art.

[0677] The present disclosure particularly recognizes that the 5'-terminal structure of oligonucleotides, optionally in combination with additional features according to the present disclosure, can provide unexpected advantages. In some embodiments, the present disclosure provides a 5'-terminal group (corresponding to the 5'-HO-CH of ribose present in natural RNA (or deoxyribose present in natural DNA)), which can surprisingly improve one or more properties and / or activities of the oligonucleotide (e.g., stability, activity, manufacturing cost, etc.). 2- ) which can surprisingly improve one or more properties and / or activities of the oligonucleotide (e.g., stability, activity, manufacturing cost, etc.).

[0678] In some embodiments, the 5'-OH groups of the provided oligonucleotides are unmodified, i.e., they exist as free -OH. In some embodiments, the 5'-terminal group is 5'-HO-CH2-. The present disclosure particularly demonstrates that the provided oligonucleotides having free 5'-OH groups can achieve properties and / or activities comparable to those of otherwise identical oligonucleotides comprising a 5'-phosphate (or its derivative) group (e.g., stability, RNAi activity when used as an ss-RNAi agent, etc.), despite reports in the literature that certain activities (e.g., RNAi activity) require the presence of a 5'-phosphate group.

[0679] In some embodiments, the 5'-terminal group does not contain a phosphorus atom. In some embodiments, the 5'-terminal group does not contain a phosphate group or its derivatives or bioisosteres. In some embodiments, the 5'-terminal group does not contain an acidic group. In some embodiments, the 5'-terminal group does not contain a carboxyl group. In some embodiments, the 5'-terminal group does not contain a phosphorus atom or a carboxyl group. In some embodiments, the 5'-terminal group is 5'-HO-CH 2-Among other things, the present disclosure demonstrates that oligonucleotides provided without a 5'-phosphate or its derivatives or bioisosteres can surprisingly achieve comparable activity to oligonucleotides having a 5'-phosphate but otherwise identical, such as activity in knocking down the mRNA level of a target gene via the RNAi pathway.

[0680] In some embodiments, the 5'-nucleoside unit of the provided oligonucleotide (which includes the sugar and nucleobase moieties but not the internucleoside linkage between the 5'-nucleoside unit and the second nucleoside unit from the 5'-end) does not contain a phosphate group or its derivatives or bioisosteres. In some embodiments, the 5'-nucleoside unit does not contain a phosphorus atom. In some embodiments, the 5'-nucleoside does not contain an acidic group. In some embodiments, the 5'-nucleoside unit does not contain a -COOH group or its salt form.

[0681] In some embodiments, the 5'-terminal group is or contains a phosphate group or its derivatives or bioisosteres. In some embodiments, the 5'-nucleoside unit contains a 5'-group that is a phosphate group or its derivatives or bioisosteres. As is known to those of ordinary skill in the art, many such groups are known in the art and can be used in accordance with the present disclosure.

[0682] In some embodiments, the 5'-terminal group is -CH2-O-P(O)(OH)-(OH) or its salt form. In some embodiments, the provided 5'-nucleoside unit has the structure or its salt form. In some embodiments, the provided 5'-nucleoside unit has the structure or its salt form. In some embodiments, X is O. In some embodiments, X is S. In some embodiments, R E is -(R)-CH(CH3)-O-P(O)(OH)-S-H or its salt form. In some embodiments, R E is -(R)-CH(CH3)-O-P(O)(OH)-O-H or its salt form. In some embodiments, R E is -(S)-CH(CH3)-O-P(O)(OH)-S-H or its salt form. In some embodiments, R E is -(S)-CH(CH3)-O-P(O)(OH)-O-H or its salt form. In some embodiments, the provided 5'-nucleoside unit has the structure or its salt form. In some embodiments, the provided 5'-nucleoside unit has the structure or its salt form.

[0683] As will be readily appreciated by one of ordinary skill in the art, the provided compounds, such as oligonucleotides or portions thereof, such as 5'-terminal structures of oligonucleotides, internucleotide linkages, etc., may exist, in some pH (e.g., physiological pH) due to, for example, one or more acidic and / or basic moieties therein, in part (sometimes predominantly) as one or more of their salt forms. In some embodiments, the provided 5'-nucleoside units may exist, in part (sometimes predominantly), as one or more of their salt forms. For example, depending on the pH, may exist as or any combination thereof. Unless otherwise expressly indicated, when listing the provided compounds or structures, all salt forms are included.

[0684] In some embodiments, R E is -L-P(O)(XR)2 or a salt form thereof. In some embodiments, R E is -L-P(O)(XR)2 or a salt form thereof, where each X is independently -O-, -S-, or a covalent bond. In some embodiments, R E is -L-P(O)(OR)2 or a salt form thereof. In some embodiments, R E is -L-P(O)(OR)(SR) or a salt form thereof. In some embodiments, R E is -L-P(O)(OR)(R) or a salt form thereof. In some embodiments, L is a covalent bond or a divalent, optionally substituted, straight-chain or branched C 1-6 aliphatic, where one or more methylene units are optionally and independently substituted with -O-, -S-, or -N(R')-. In some embodiments, R E is -L-R 5s . In some embodiments, R E is -X-L-R. In some embodiments, R E is In some embodiments, R E is -C(R) 2- . In some embodiments, X is -O-. In some embodiments, X is -S-. In some embodiments, X is -N(R)-. In some embodiments, L comprises an optionally substituted, divalent or polyvalent group. In some embodiments, L comprises an optionally substituted group. In some embodiments, L comprises group. In some embodiments, R is independently -H or an optionally substituted group selected from C 1-10 alkyl, C 1-10 allyl, and C 6-14 aryl. In some embodiments, R is -H. In some embodiments, RE is optionally substituted In some embodiments, R E is

[0685] Many phosphate ester derivatives and / or bioisosteres and 5'-nucleoside units are described in the literature and can be used in accordance with the present disclosure. For example, some such structures are described in, e.g., US2016-0194349; US2016-0186175; US20130323836, etc. In some embodiments, the 5'-terminal group R E or 5'-nucleoside units are described in, e.g., Allerson et al. 2005 J. Med. Chem. 48:901-04; Lima et al. 2012 Cell 150:883-894; Prakash et al. 2015 Nucl. Acids Res. 43:2993-3011; and / or Prakash et al. 2016 Bioorg. Med. Chem. Lett. 26:26:2817-2820, such as T-VP, T-PO, etc.

[0686] Bridged morpholino and cyclohexenyl nucleotides and nucleosides are described in, e.g., U.S. Patent Application Publication 2016-0186175, which can be used in accordance with the present disclosure.

[0687] Exemplary embodiments of the variables are described extensively in the present disclosure. For structures having two or more variables, unless otherwise specified, each variable can independently be any of the embodiments described herein.

[0688] In some embodiments, an APOC3 oligonucleotide capable of directing the reduction of the expression and / or level of a target gene or its gene product can comprise any 5'-end described herein or known in the art.

[0689] In some embodiments, an APOC3 oligonucleotide, an APOC3 oligonucleotide that directs RNA interference, or an APOC3 oligonucleotide that directs RNase H-mediated knockdown can comprise any 5'-end described herein or known in the art.

[0690] In some embodiments, an APOC3 oligonucleotide, an APOC3 oligonucleotide that directs RNase H-mediated knockdown, or an APOC3 oligonucleotide that simultaneously directs RNA interference and RNase H-mediated knockdown can comprise any 5'-end described herein or known in the art.

[0691] In some embodiments, the 5'-end of the provided single-stranded RNAi agent comprises a phosphorus-containing moiety (e.g., the 5'-end comprises phosphorus). Non-limiting examples of ssRNAi formats in which the 5'-end comprises a phosphorus-containing moiety include Formats 1-15, 20-21, 23-31, 80-82, 92-95, 97-102, and 104-107 of Figure 1.

[0692] In some embodiments, the 5'-end of the provided single-stranded RNAi agent does not comprise a phosphorus-containing moiety (e.g., the 5'-end comprises phosphorus). Non-limiting examples of ssRNAi formats in which the 5'-end does not comprise a phosphorus-containing moiety include Formats 16-19, 22, 32-79, 83-91, 96, and 103 of Figure 1.

[0693] In some embodiments, the 5'-end of the provided single-stranded RNAi agent has a moiety comprising a phosphoester (such as a phosphodiester, phosphorothioate, dithiophosphate, H-phosphonate), or other moiety similar or identical to the internucleotide linkage comprising a phosphoester. In some embodiments, the 5'-end of the provided single-stranded RNAi agent has a moiety comprising a phosphoester but not a phosphodiester; in some embodiments, such a moiety is referred to as a phosphoester mimic, modified phosphoester, or phosphoester analogue.

[0694] In some embodiments, the 5'-end of the provided single-stranded RNAi agent does not have a moiety comprising a phosphoester.

[0695] In some embodiments of the single-stranded RNAi agent, the 5'-end structure has a structure selected from the following: 5'-(R)-MeOH T, 5'-(R)-Me PO T, 5'-(R)-Me PS T, 5'-(R)-Me PH T, 5'-(S)-Me OH T, 5'-(S)-Me POT, 5'-(S)-Me PS T, and 5'-(S)-PH T.

[0696] In some embodiments of the provided single-stranded RNAi agent, it comprises a phosphorus-containing moiety at the 5'-end structure, and the 5'-end structure is represented by a structure selected from the following: Formula IV-a (Mod022, also known as C3 PO and n-propyl), IV-b (Mod022*), IV-c (POMod023*), IV-d (PSMod023*), and IV-e (PHMod023*):

[0697]

[0698] In some embodiments of the provided single-stranded RNAi agents, they comprise a phosphorus-containing moiety at the 5'-terminal structure, and the 5'-terminal structure is represented by a structure selected from the following: the structure of formula IV-f (also known as n-propyl C3 PO or Mod022):

[0699]

[0700] where 5' represents the point of attachment to the 5'-carbon of the sugar.

[0701] In some embodiments of the provided single-stranded RNAi agents, they comprise a phosphorus-containing moiety at the 5'-terminal structure, and the 5'-terminal structure is represented by a structure selected from the following: (also known as C3PS or Mod022*) structure:

[0702]

[0703] where 5' represents the point of attachment to the 5'-carbon of the sugar (e.g., N1).

[0704] In some embodiments of the provided single-stranded RNAi agents, they comprise a phosphorus-containing moiety at the 5'-terminal structure, and the 5'-terminal structure is represented by a structure selected from the following: the structure of formula IV-g (also known as dimethyl C3 or C3 dimethyl PS or Mod023*):

[0705]

[0706] where 5' represents the point of attachment to the 5'-carbon of the sugar (e.g., N1).

[0707] In some embodiments, the single-stranded RNAi agent comprises a 5'-terminal structure selected from any one of the following: PO (phosphodiester), formula IV-h; PH (H-phosphonate), formula IV-i; and PS (phosphorothioate), formula IV-j:

[0708]

[0709] In some embodiments of the provided single-stranded RNAi agents, they comprise a phosphorus-containing moiety at the 5'-terminal structure, and the 5'-terminal structure is represented by a structure selected from any one of the following:

[0710]

[0711] where 5' represents the point of attachment to the 5'-carbon of the sugar (e.g., N1).

[0712] In some embodiments, P in any one of formulas IV-a to IV-j is stereorandom or stereodefined as in the Sp or Rp configuration.

[0713] In some embodiments, the 5′-terminal structure is selected from any of the following: phosphate, phosphate analog, 5′-monophosphate ((HO)2(O)P-O-5′), 5′-diphosphate ((HO)2(O)P-O-P(HO)(O)-O-5′), 5′-triphosphate ((HO)2(O)P-O-(HO)(O)P-O-P(HO)(O)-O-5′), 5′-guanosine cap (7-methylated or non-methylated) (7m-G-O-5′-(HO)(O)P-O-(HO)(O)P-O-P(HO)(O)-O-5′), 5′-adenosine cap (Appp), and any modified or unmodified nucleotide cap structure (N-O-5′-(HO)(O)P-O-(HO)(O)P-O-P(HO)(O)-O-5′), 5′-monothiophosphate (thiophosphate; (HO)2(S)P-O-5′), 5′-monodithiophosphate (dithiophosphate; (HO)(HS)(S)P-O-5′), 5′-thiophosphate ((HO)2(O)P-S-5′); any additional combination of oxygen / sulfur-substituted monophosphates, diphosphates, and triphosphates (e.g., 5′-α-thiotriphosphate, 5′-γ-thiotriphosphate, etc.), 5′-aminophosphate ((HO)2(O)P-NH-5′, (HO)(NH2)(O)P-O-5′), 5′-alkylphosphonate (R = alkyl = methyl, ethyl, isopropyl, propyl, etc., e.g., RP(OH)(O)-O-5′-, (OH)2(O)P-5′-CH2-), 5′-alkyl ether phosphonate (R = alkyl ether = methoxymethyl (MeOCH2-), ethoxymethyl, etc., e.g., RP(OH)(O)-O-5′-).

[0714] In some embodiments, 5′-terminals containing a phosphorus-containing moiety may have particular advantages because single-stranded RNAi agents containing them may be more active in RNA interference.

[0715] In some embodiments, the 5′-terminal structure has the following structure: 5′-nucleotide or modified 5′-nucleotide, 5′-nucleotide analog, 5′-nucleoside or modified 5′-nucleoside or 5′-nucleoside analog.

[0716] In some embodiments, the 5′-end structure has the structure of any of the following: 5′-guanosine cap, 5′-adenosine cap, 5′-monothiophosphate, 5′-monodithiophosphate, 5′-thiophosphate, 5′-aminophosphate, 5′-alkylphosphonate, and 5′-alkyl ether phosphonate; 5′-monophosphate, 5′-diphosphate, and 5′-triphosphate; 5′-triphosphate; monophosphate, diphosphate, or triphosphate, wherein at least one oxygen atom in the monophosphate, diphosphate, or triphosphate is replaced by a sulfur atom; 5′-α-thiotriphosphate and 5′-γ-thiotriphosphate; alkylphosphonate; the alkylphosphonate has the formula: RP(OH)(O)--O-5′ or (OH)2(O)P-5′-CH2--, wherein R is a C1-C3 alkyl; alkyl ether phosphonate; or an alkyl ether phosphonate having the following formula: RP(OH)(O)--O-5′, wherein R is an alkyl ether.

[0717] A variety of 5′-nucleosides are described, for example, in U.S. Patent Application 14 / 959,714 published as US 2016-0194349 A1; U.S. Patent Application 14 / 983,907 published as US 2016-0186175 A1; or U.S. Patent Application 13 / 696,796 published as US 20130323836.

[0718] In some embodiments, the 5′-terminal structure is vinyl phosphonate.

[0719] In certain embodiments, an oligomeric compound is provided, wherein the 5′-terminal compound has the formula VIII-c, wherein G is F, OCH3, or O(CH2)2--OCH3.

[0720] In some embodiments, the 5′-end structure has a structure selected from the following: 5′-(R)-Me OH T, 5′-(R)-Me PO T, 5′-(R)-Me PS T, 5′-(R)-Me PH T, 5′-(S)-Me OH T, 5′-(S)-Me PO T, 5′-(S)-MePS T, and 5′-(S)-PH T.

[0721] In some embodiments, the 5′-end structure has the structure of 5′-(R)-Me OH T.

[0722] In some embodiments, the 5′-end structure has the structure of 5′-(R)-Me PO T.

[0723] In some embodiments, the 5′-end structure has the structure of 5′-(R)-Me PS T.

[0724] In some embodiments, the 5′-terminal structure has the structure of 5′-(R)-Me PH T.

[0725] In some embodiments, the 5′-terminal structure has the structure of 5′-(S)-Me OH T.

[0726] In some embodiments, the 5′-terminal structure has the structure of 5′-(S)-Me PO T.

[0727] In some embodiments, the 5′-terminal structure has the structure of 5′-(S)-Me PS T.

[0728] In some embodiments of the single-stranded RNAi agent, the 5′-terminal structure has the structure of 5′-(R)-Me PO T.

[0729] In some embodiments of the single-stranded RNAi agent, the 5′-terminal structure has the structure of 5′-(R)-Me PS T.

[0730] In some embodiments of the single-stranded RNAi agent, the 5′-terminal structure has the structure of 5′-(R)-Me PH T.

[0731] In some embodiments of the single-stranded RNAi agent, the 5′-terminal structure has the structure of 5′-(S)-Me OH T.

[0732] In some embodiments of the single-stranded RNAi agent, the 5′-terminal structure has the structure of 5′-(S)-Me PO T.

[0733] In some embodiments of the single-stranded RNAi agent, the 5′-terminal structure has the structure of 5′-(S)-Me PS T.

[0734] In some embodiments of the single-stranded RNAi agent, the 5′-terminal structure has the structure of 5′-(S)-Me PH T.

[0735] In some embodiments, the 5′-terminal structure has the structure of 5′-(S)-Me PH T. Additionally, some references, such as EP 1520022 B1, paragraph 6, have reported that a 5′-phosphate is required on the target complementary strand (e.g., the antisense strand) of the siRNA duplex for RISC activity. Column 2 of U.S. Patent No. 8,729,036 also indicates that a 5′-phosphate has been reported to be essential for RNA interference. Column 3 of U.S. Patent No. 8,729,036 further reports that a 5′-phosphate is required for single-stranded antisense siRNAs to trigger RNAi in HeLa S100 extracts. However, the present disclosure has demonstrated that various single-stranded RNAi agents that do not contain a 5′-phosphate are capable of directing RNA interference.

[0736] In some embodiments, the 5′-end comprises a phosphate-containing moiety (such as T-VP or T-PO) or any other suitable RNAi agent 5′-end compound as described in, for example, Allerson et al. 2005 J. Med. Chem. 48:901-04; ...

Claims

1. A compound having the formula O1: Y 1 -L 1 -(Z 10 ) za O1 or a pharmaceutically acceptable salt of said compound, wherein Y 1 is an oligonucleotide targeting APOC3; za is 1, 2 or 3; and L 1 is a compound of formula L11, L12, L13, L43, L44, L45, L46, L47, L48, L49, L50, L51, L52, L53 or L54, wherein the attachment sites to Y 1 and Z 10 are represented as: wherein each T 1 independently does not exist or is an alkylene, alkenylene or alkynylene group, wherein one or more -CH2- groups of said alkylene, alkenylene or alkynylene group may each independently be replaced by a heteroatom group independently selected from -O-, -S- and -N(R 49 )-, wherein said heteroatom group is separated by at least 2 carbon atoms; Each Q 1 independently does not exist or is -C(O)-, -C(O)-NR 49 -, -NR 49 -, -C(O)-, -O-C(O)-NR 49 -, -NR 49 -, -C(O)-O-, -CH2-, -NR 49 C(O)NR 49 -, a divalent heteroaryl group or a heteroatomic group selected from -O-, -S-, -S-S-, -S(O)-, -S(O)2- and -NR 49 -, wherein at least two carbon atoms separate the heteroatomic groups -O-, -S-, -S-S-, -S(O)-, -S(O)2- and -NR 49 - from any other heteroatomic group, or a structure of the following formula: wherein R 53 is -O or -NH-; and R 54 is -O or -S; Each R 49 is independently -H, -(C1-C 20 )alkyl or -(C3-C6)cycloalkyl, wherein one to six -CH2- groups of said alkyl or cycloalkyl separated by at least two carbon atoms may be replaced by -O-, -S- or -N(R 49a ), and -CH3 of said alkyl may be replaced by a heteroatom group selected from -N(R 49a )2, -OR 49a and -S(R 49a ), wherein said heteroatom groups are separated by at least 2 carbon atoms; and wherein said alkyl and cycloalkyl may be substituted by halogen atoms; and wherein each R 49a is independently -H, -(C1-C6)alkyl or -(C3-C6)cycloalkyl; R 53 is -O or -NH; R 54 is -O or -S; Each n is independently 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39 or 40; provided that if n is greater than 0, then each (T 1 -Q 1 -T 1 -Q 1 ) of each T 1 and each Q 1 ; and Each Z 10 is independently a compound of formula Z12, Z13, Z14, Z15, Z16, Z17, Z18, Z19, Z20 or Z21 or a geometric or positional isomer thereof, wherein the attachment site to L 1 is represented as: where each R 46 is independently -CN, -CH2-CN, -C≡CH, -CH2-N3, -CH2-NH2, -CH2-N(R 52 )-S(O)2-R 51 , -CH2-CO2H, -CO2H, -CH2-OH, -CH2-SH, -CH=CH-R 51 , -CH2-R 51 , -CH2-S-R 51 , -CH2-N(R 52 )-R 51 , -CH2-N(R 52 )-C(O)-R 51 , -CH2-N(R 52 )-C(O)-O-R 51 , -CH2-N(R 52 )-C(O)-N(R 52 )-R 51 , -CH2-O-R 51 , -CH2-O-C(O)-R 51 , -CH2-O-C(O)-N(R 52 )-R 51 , -CH2-O-C(O)-O-R 51 , -CH2-S(O)-R 51 , -CH2-S(O)2-R 51 , -CH2-S(O)2-N(R 52 )-R 51 , -C(O)-NH2, -C(O)-O-R 51 , -C(O)-N(R 52 )-R 51 or an aryl or heteroaryl group, where the aryl or heteroaryl group is optionally substituted with R 51 ; Each R 47 is independently -OH, -N3, -N(R 48 )2, -N(R 48 )-C(O)-R 48 , -N(R 48 )-C(O)-N(R 48 )2, -N(R 48 )-C(O)-OR 48 , -N(R 48 )-S(O)2-R 48 , tetrazole or triazole, wherein the tetrazole and triazole are optionally substituted by R 48 ; Each R 48 is independently -H, -(C1-C5)alkyl, halogen-substituted (C1-C5)alkyl, halogen-substituted -(C3-C6)cycloalkyl, -(C1-C5)alkenyl, -(C1-C5)alkynyl, halogen-substituted -(C1-C5)alkenyl, halogen-substituted -(C1-C5)alkynyl or -(C3-C6)cycloalkyl, wherein the -CH2- groups of said alkyl or cycloalkyl may each independently be replaced by a heteroatom group selected from -O-, -S- and -N(R 52 ), and the -CH3 of said alkyl may each independently be replaced by a heteroatom group selected from -N(R 52 )2, -OR 52 and -S(R 52 ), wherein said heteroatom groups are separated by at least 2 carbon atoms; Each R 51 is independently -H, -(C3-C 20 ) cycloalkyl, -(C1-C 60 ) alkenyl, -(C1-C 60 ) alkynyl or -(C1-C 60 ) alkyl, wherein one to six -CH2- groups of the cycloalkyl or one to twenty -CH2- groups of the alkyl may each independently be replaced by a heteroatom independently selected from -O-, -S- or -N(R 49 )-, wherein the heteroatoms are separated by at least two carbon atoms, and the -CH3 of the alkyl may each independently be replaced by a heteroatom group selected from -N(R 49 )2, -OR 49 and -S(R 49 ), wherein the heteroatom groups are separated by at least 2 carbon atoms, and wherein the alkyl, alkenyl, alkynyl and cycloalkyl may be substituted by halogen atoms; and Each R 52 is independently -H, -(C1-C 20 )alkyl, -(C1-C 20 )alkenyl, -(C1-C 20 )alkynyl or -(C3-C6)cycloalkyl, wherein one to six -CH2- groups of said alkyl or cycloalkyl separated by at least two carbon atoms may each independently be replaced by a heteroatom independently selected from -O-, -S- or -N(R 49 ), and the -CH3 of said alkyl may each independently be replaced by a heteroatom group selected from -N(R 49 )2, -OR 49 and -S(R 49 ), wherein said heteroatom groups are separated by at least 2 carbon atoms; and wherein said alkyl, alkenyl, alkynyl and cycloalkyl may be substituted by a halogen atom.

2. The compound according to any one of the preceding claims, wherein Y 1 comprises at least 15 bases.

3. The compound according to any one of the preceding claims, wherein Y 1 has a base sequence that comprises or is the base sequence of any of the APOC3 oligonucleotides listed in Table 1A, or Y 1 has a base sequence that comprises 15 consecutive bases of the sequence of any of the APOC3 oligonucleotides listed in Table 1A.

4. A compound according to any one of the preceding claims, wherein Y 1 comprises at least one internucleoside phosphodiester bond.

5. A compound according to any one of the preceding claims, wherein Y 1 comprises at least one chirally controlled modified internucleoside linkage.

6. A compound as claimed in any one of the preceding claims, Y 1 comprising at least 1 chirality-controlled modified internucleoside linkage, which is a chirality-controlled phosphorothioate.

7. A compound according to any one of the preceding claims, wherein Y 1 comprises at least one chirality-controlled modified internucleoside linkage, which is a chirality-controlled phosphorothioate in the Sp configuration.

8. A compound according to any one of the preceding claims, wherein Y 1 comprises at least 1 chirality-controlled modified internucleoside linkage which is a chirality-controlled phosphorothioate in the Rp configuration.

9. A compound according to any one of the preceding claims, wherein Y 1 , wherein the chirally controlled modified internucleoside linkage or chirally controlled phosphorothioate comprises a phosphorus chiral center having at least 70% diastereomeric purity in the composition.

10. A compound according to any one of the preceding claims, wherein Y 1 , wherein the chirally controlled modified internucleoside linkage or chirally controlled phosphorothioate comprises a phosphorus chiral center having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.5% diastereomeric purity.

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